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1
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Miolo G, Canil G, Polano M, Dal Bo M, Mondello A, Palumbo A, Puglisi F, Corona G. Novel De Novo BRCA2 Variant in an Early-Onset Ovarian Cancer Reveals a Unique Tumor Evolution Pathway. Int J Mol Sci 2025; 26:2295. [PMID: 40076915 PMCID: PMC11899998 DOI: 10.3390/ijms26052295] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2025] [Revised: 02/27/2025] [Accepted: 03/01/2025] [Indexed: 03/14/2025] Open
Abstract
Ovarian cancer (OC) is a highly heterogeneous malignancy, often characterized by complex genomic alterations that drive tumor progression and therapy resistance. In this paper, we report a novel de novo BRCA2 germline variant NM_000059.3:c.(8693_8695delinsGT) associated with early-onset OC that featured two regions with differential MMR (Mismatch Repair) gene expression. To date, only six cases of de novo BRCA2 variants have been reported, none of which were associated with early-onset high-grade serous OC. The immunohistochemical analysis of MMR genes revealed two distinct tumor areas, separated by a clear topographic boundary, with the heterogeneous expression of MLH1 and PMS2 proteins. Seventy-five percent of the tumor tissue showed positivity, while the remaining 25% exhibited a complete absence of expression, underscoring the spatial variability in MMR gene expression within the tumor. Integrated comparative spatial genomic profiling identified several tumor features associated with the genetic variant as regions of loss of heterozygosity (LOH) that involved BRCA2 and MLH1 genes, along with a significantly higher mutational tumor burden in the tumor area that lacked MLH1 and PMS2 expression, indicating its further molecular evolution. The following variants were acquired: c.6572C>T in NOTCH2, c.1852C>T in BCL6, c.191A>T in INHBA, c.749C>T in CUX1, c.898C>A in FANCG, and c.1712G>C in KDM6A. Integrated comparative spatial proteomic profiles revealed defects in the DNA repair pathways, as well as significant alterations in the extracellular matrix (ECM). The differential expression of proteins involved in DNA repair, particularly those associated with MMR and Base Excision Repair (BER), highlights the critical role of defective repair mechanisms in driving genomic instability. Furthermore, ECM components, such as collagen isoforms, Fibrillin-1, EMILIN-1, Prolargin, and Lumican, were found to be highly expressed in the MLH1/PMS2-deficient tumor area, suggesting a connection between DNA repair deficiencies, ECM remodeling, and tumor progression. Thus, the identification of the BRCA2 variant sheds light on the poorly understood interplay between DNA repair deficiencies and ECM remodeling in OC, providing new insights into their dual role in shaping tumor evolution and suggesting potential targets for novel therapeutic strategies.
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Affiliation(s)
- Gianmaria Miolo
- Medical Oncology and Cancer Prevention Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 33081 Aviano, Italy; (G.M.); (F.P.)
| | - Giovanni Canil
- Immunopathology and Cancer Biomarkers Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 33081 Aviano, Italy;
| | - Maurizio Polano
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 33081 Aviano, Italy; (M.P.); (M.D.B.); (A.M.)
| | - Michele Dal Bo
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 33081 Aviano, Italy; (M.P.); (M.D.B.); (A.M.)
| | - Alessia Mondello
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 33081 Aviano, Italy; (M.P.); (M.D.B.); (A.M.)
| | - Antonio Palumbo
- Pathology Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 33081 Aviano, Italy;
| | - Fabio Puglisi
- Medical Oncology and Cancer Prevention Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 33081 Aviano, Italy; (G.M.); (F.P.)
- Department of Medicine, University of Udine, 33100 Udine, Italy
| | - Giuseppe Corona
- Immunopathology and Cancer Biomarkers Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 33081 Aviano, Italy;
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Humphries EM, Hains PG, Robinson PJ. Overlap of Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Matched Tissues for Proteomics and Phosphoproteomics. ACS OMEGA 2025; 10:6891-6900. [PMID: 40028131 PMCID: PMC11865994 DOI: 10.1021/acsomega.4c09289] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/10/2024] [Revised: 01/08/2025] [Accepted: 01/15/2025] [Indexed: 03/05/2025]
Abstract
Many liquid chromatography-mass spectrometry (LC-MS) studies have compared formalin-fixed paraffin-embedded (FFPE) tissues with matched fresh-frozen (FF) tissues to examine the effect of preservation techniques on the proteome; however, few studies have included the phosphoproteome. A high degree of overlap and correlation between the two preservation techniques would demonstrate the importance of FFPE tissues as a valuable biomedical resource. Our aim was to quantitatively compare the proteome and phosphoproteome of matched FFPE and FF tissues using data-independent acquisition LC-MS. Four organs from three rats were cut in half to produce matched FFPE and FF tissue pairs. Excellent overlaps of 85-97% for the proteome and 82-98% for the phosphoproteome were observed, depending on the organ type, between the two preservation techniques. Most of the unique identifications were found in FF with less than 0.3% being unique to FFPE tissues. Strong agreement between FFPE and FF matched tissue pairs was observed with Pearson correlation coefficients of 0.93-0.97 and 0.79-0.87 for the proteome and phosphoproteome, respectively. Digestion efficiency was slightly higher in FFPE (92-94%) than in FF tissues (86-89%), and a search of a data subset for formaldehyde induced chemical modifications revealed that only 0.05% of precursors were unique to FFPE tissues. This suggests that with quality sample preparation methods it is not necessary to include formaldehyde induced chemical modifications when analyzing FFPE tissues. We attribute the lower number of identifications in FFPE tissues to inaccurate peptide quantitation, which resulted in a lower MS peptide load and tryptic peptide enrichment load. Our results demonstrate that both proteomic and phosphoproteomic analyses of FFPE and FF tissues are highly comparable and highlight the suitability of FFPE tissues for both proteomic and phosphoproteomic analysis.
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Affiliation(s)
- Erin M. Humphries
- ProCan, Children’s
Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia
| | - Peter G. Hains
- ProCan, Children’s
Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia
| | - Phillip J. Robinson
- ProCan, Children’s
Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia
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3
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Lama S, Muneer F, America AH, Kuktaite R. Polymeric Gluten Proteins as Climate-Resilient Markers of Quality: Can LC-MS/MS Provide Valuable Information about Spring Wheat Grown in Diverse Climates? JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2025; 73:1844-1854. [PMID: 39786935 PMCID: PMC11760153 DOI: 10.1021/acs.jafc.4c10789] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/04/2024] [Revised: 12/20/2024] [Accepted: 12/25/2024] [Indexed: 01/12/2025]
Abstract
In this study, the impact of the varying environments, wet-cool (2017), dry-hot (2018), and fluctuating (2019), on two spring wheat genotypes, Diskett and Bumble, grown in field conditions in southern Sweden was studied. From harvested grains, polymeric gluten proteins were fractionated and collected using SE-HPLC and then analyzed with LC-MS/MS. Proteins and peptides identified through searches against the protein sequences ofTriticum aestivum (taxon 4565) from the UniProtKB database showed 7 high molecular weight glutenin subunits (HMW-GS) and 24 low molecular weight glutenin subunits (LMW-GS) with different enrichment levels for both genotypes. Glu-B1 for HMW-GS and Glu D3 and m- and s-types for LMW-GS were dominated in both genotypes, and a small proportion of α-, γ-, and ω-gliadins were also present. A minor variation in HMW-GS and LMW-GS compositions was observed between the years, while small amounts of heat shock proteins were identified under the "dry-hot" period for Diskett. In conclusion, Diskett showed more stable and climate-resistant protein patterns in the studied varying climate as compared to Bumble. The study highlights the use of proteomics and LC-MS/MS for differentiation of wheat genotypes, although it shows low sensitivity in measuring the diverse environment impact on the polymeric proteins.
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Affiliation(s)
- Sbatie Lama
- Department
of Plant Breeding, Swedish University of
Agricultural Sciences, Box 190, Lomma SE-23422, Sweden
- Agricultural
University of Iceland, H66H+9R9, Hvanneyrabraut, Hvanneyri 311, Iceland
| | - Faraz Muneer
- Department
of Plant Breeding, Swedish University of
Agricultural Sciences, Box 190, Lomma SE-23422, Sweden
| | - Antoine H.P. America
- Wageningen
Plant Research, Wageningen University and
Research, Wageningen 6708 PB, The Netherlands
| | - Ramune Kuktaite
- Department
of Plant Breeding, Swedish University of
Agricultural Sciences, Box 190, Lomma SE-23422, Sweden
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4
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Perez JM, Duda JM, Ryu J, Shetty M, Mehta S, Jagtap PD, Nelson AC, Winterhoff B, Griffin TJ, Starr TK, Thomas SN. Investigating proteogenomic divergence in patient-derived xenograft models of ovarian cancer. Sci Rep 2025; 15:813. [PMID: 39755759 PMCID: PMC11700199 DOI: 10.1038/s41598-024-84874-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2024] [Accepted: 12/27/2024] [Indexed: 01/06/2025] Open
Abstract
Within ovarian cancer research, patient-derived xenograft (PDX) models recapitulate histologic features and genomic aberrations found in original tumors. However, conflicting data from published studies have demonstrated significant transcriptional differences between PDXs and original tumors, challenging the fidelity of these models. We employed a quantitative mass spectrometry-based proteomic approach coupled with generation of patient-specific databases using RNA-seq data to investigate the proteogenomic landscape of serially-passaged PDX models established from two patients with distinct subtypes of ovarian cancer. We demonstrate that the utilization of patient-specific databases guided by transcriptional profiles increases the depth of human protein identification in PDX models. Our data show that human proteomes of serially passaged PDXs differ significantly from their patient-derived tumor of origin. Analysis of differentially abundant proteins revealed enrichment of distinct biological pathways with major downregulated processes including extracellular matrix organization and the immune system. Finally, we investigated the relative abundances of ovarian cancer-related proteins identified from the Cancer Gene Census across serially passaged PDXs, and found their protein levels to be unstable across PDX models. Our findings highlight features of distinct and dynamic proteomes of serially-passaged PDX models of ovarian cancer.
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Affiliation(s)
- Jesenia M Perez
- Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota School of Medicine, Minneapolis, MN, 55455, USA
| | - Jolene M Duda
- Biochemistry, Molecular Biology and Biophysics, University of Minnesota School of Medicine, Minneapolis, MN, 55455, USA
| | - Joohyun Ryu
- Department of Laboratory Medicine and Pathology, University of Minnesota School of Medicine, 420 Delaware St SE, MMC 609, Minneapolis, MN, 55455, USA
| | - Mihir Shetty
- Masonic Cancer Center and Department of Obstetrics, Gynecology and Women's Health, University of Minnesota, Minneapolis, MN, 55455, USA
| | - Subina Mehta
- Biochemistry, Molecular Biology and Biophysics, University of Minnesota School of Medicine, Minneapolis, MN, 55455, USA
| | - Pratik D Jagtap
- Biochemistry, Molecular Biology and Biophysics, University of Minnesota School of Medicine, Minneapolis, MN, 55455, USA
| | - Andrew C Nelson
- Department of Laboratory Medicine and Pathology, University of Minnesota School of Medicine, 420 Delaware St SE, MMC 609, Minneapolis, MN, 55455, USA
| | - Boris Winterhoff
- Masonic Cancer Center and Department of Obstetrics, Gynecology and Women's Health, University of Minnesota, Minneapolis, MN, 55455, USA
| | - Timothy J Griffin
- Biochemistry, Molecular Biology and Biophysics, University of Minnesota School of Medicine, Minneapolis, MN, 55455, USA
| | - Timothy K Starr
- Masonic Cancer Center and Department of Obstetrics, Gynecology and Women's Health, University of Minnesota, Minneapolis, MN, 55455, USA
| | - Stefani N Thomas
- Department of Laboratory Medicine and Pathology, University of Minnesota School of Medicine, 420 Delaware St SE, MMC 609, Minneapolis, MN, 55455, USA.
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5
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Nordmann TM, Mund A, Mann M. A new understanding of tissue biology from MS-based proteomics at single-cell resolution. Nat Methods 2024; 21:2220-2222. [PMID: 39643675 DOI: 10.1038/s41592-024-02541-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/09/2024]
Affiliation(s)
- Thierry M Nordmann
- Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
| | - Andreas Mund
- Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
- OmicVision Biosciences, BioInnovation Institute, Copenhagen, Denmark.
| | - Matthias Mann
- Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
- Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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6
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Humphries EM, Loudon C, Craft GE, Hains PG, Robinson PJ. Quantitative Comparison of Deparaffinization, Rehydration, and Extraction Methods for FFPE Tissue Proteomics and Phosphoproteomics. Anal Chem 2024; 96:13358-13370. [PMID: 39102789 DOI: 10.1021/acs.analchem.3c04479] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/07/2024]
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissues are suitable for proteomic and phosphoproteomic biomarker studies by data-independent acquisition mass spectrometry. The choice of the sample preparation method influences the number, intensity, and reproducibility of identifications. By comparing four deparaffinization and rehydration methods, including heptane, histolene, SubX, and xylene, we found that heptane and methanol produced the lowest coefficients of variation (CVs). Using this, five extraction methods from the literature were modified and evaluated for their performance using kidney, leg muscle, lung, and testicular rat organs. All methods performed well, except for SP3 due to insufficient tissue lysis. Heat n' Beat was the fastest and most reproducible method with the highest digestion efficiency and lowest CVs. S-Trap produced the highest peptide yield, while TFE produced the best phosphopeptide enrichment efficiency. The quantitation of FFPE-derived peptides remains an ongoing challenge with bias in UV and fluorescence assays across methods, most notably in SPEED. Functional enrichment analysis demonstrated that each method favored extracting some gene ontology cellular components over others including chromosome, cytoplasmic, cytoskeleton, endoplasmic reticulum, membrane, mitochondrion, and nucleoplasm protein groups. The outcome is a set of recommendations for choosing the most appropriate method for different settings.
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Affiliation(s)
- Erin M Humphries
- ProCan, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, New South Wales 2145, Australia
| | - Clare Loudon
- ProCan, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, New South Wales 2145, Australia
| | - George E Craft
- ProCan, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, New South Wales 2145, Australia
| | - Peter G Hains
- ProCan, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, New South Wales 2145, Australia
| | - Phillip J Robinson
- ProCan, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, New South Wales 2145, Australia
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7
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Naimy S, Sølberg JBK, Kuczek DE, Løvendorf MB, Bzorek M, Litman T, Mund A, Rahbek Gjerdrum LM, Clark RA, Mann M, Dyring-Andersen B. Comparative Quantitative Proteomic Analysis of Melanoma Subtypes, Nevus-Associated Melanoma, and Corresponding Nevi. J Invest Dermatol 2024; 144:1608-1621.e4. [PMID: 38185415 DOI: 10.1016/j.jid.2023.12.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Revised: 12/01/2023] [Accepted: 12/04/2023] [Indexed: 01/09/2024]
Abstract
A substantial part of cutaneous malignant melanomas develops from benign nevi. However, the precise molecular events driving the transformation from benign to malignant melanoma are not well-understood. We used laser microdissection and mass spectrometry to analyze the proteomes of melanoma subtypes, including superficial spreading melanomas (n = 17), nodular melanomas (n = 17), and acral melanomas (n = 15). Furthermore, we compared the proteomes of nevi cells with those of melanoma cells within the same specimens (nevus-associated melanoma (n = 14)). In total, we quantified 7935 proteins. Despite the genomic and clinical differences of the melanoma subtypes, our analysis revealed relatively similar proteomes, except for the upregulation of proteins involved in immune activation in nodular melanomas versus acral melanomas. Examining nevus-associated melanoma versus nevi, we found 1725 differentially expressed proteins (false discovery rate < 0.05). Among these proteins were 140 that overlapped with cancer hallmarks, tumor suppressors, and regulators of metabolism and cell cycle. Pathway analysis indicated aberrant activation of the phosphoinositide 3-kinase-protein kinase B-mTOR pathways and the Hippo-YAP pathway. Using a classifier, we identified six proteins capable of distinguishing melanoma from nevi samples. Our study represents a comprehensive comparative analysis of the proteome in melanoma subtypes and associated nevi, offering insights into the biological behavior of these distinct entities.
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Affiliation(s)
- Soraya Naimy
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Department of Pathology, Zealand University Hospital, Roskilde, Denmark
| | - Julie B K Sølberg
- Department of Dermatology and Allergy, Herlev and Gentofte Hospital, Copenhagen University Hospitals, Copenhagen, Denmark
| | - Dorota E Kuczek
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Marianne Bengtson Løvendorf
- Department of Dermatology and Allergy, Herlev and Gentofte Hospital, Copenhagen University Hospitals, Copenhagen, Denmark; Leo Foundation Skin Immunology Research Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Michael Bzorek
- Department of Pathology, Zealand University Hospital, Roskilde, Denmark
| | - Thomas Litman
- Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Andreas Mund
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | | | - Rachael A Clark
- Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Matthias Mann
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Beatrice Dyring-Andersen
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Department of Dermatology and Allergy, Herlev and Gentofte Hospital, Copenhagen University Hospitals, Copenhagen, Denmark; Leo Foundation Skin Immunology Research Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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8
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Thiery J, Fahrner M. Integration of proteomics in the molecular tumor board. Proteomics 2024; 24:e2300002. [PMID: 38143279 DOI: 10.1002/pmic.202300002] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2023] [Revised: 12/03/2023] [Accepted: 12/05/2023] [Indexed: 12/26/2023]
Abstract
Cancer remains one of the most complex and challenging diseases in mankind. To address the need for a personalized treatment approach for particularly complex tumor cases, molecular tumor boards (MTBs) have been initiated. MTBs are interdisciplinary teams that perform in-depth molecular diagnostics to cooperatively and interdisciplinarily advise on the best therapeutic strategy. Current molecular diagnostics are routinely performed on the transcriptomic and genomic levels, aiming to identify tumor-driving mutations. However, these approaches can only partially capture the actual phenotype and the molecular key players of tumor growth and progression. Thus, direct investigation of the expressed proteins and activated signaling pathways provide valuable complementary information on the tumor-driving molecular characteristics of the tissue. Technological advancements in mass spectrometry-based proteomics enable the robust, rapid, and sensitive detection of thousands of proteins in minimal sample amounts, paving the way for clinical proteomics and the probing of oncogenic signaling activity. Therefore, proteomics is currently being integrated into molecular diagnostics within MTBs and holds promising potential in aiding tumor classification and identifying personalized treatment strategies. This review introduces MTBs and describes current clinical proteomics, its potential in precision oncology, and highlights the benefits of multi-omic data integration.
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Affiliation(s)
- Johanna Thiery
- Institute for Surgical Pathology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Matthias Fahrner
- Institute for Surgical Pathology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- German Cancer Consortium (DKTK) and Cancer Research Center (DKFZ), Freiburg, Germany
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9
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Chamrád I, Simerský R, Lenobel R, Novák O. Exploring affinity chromatography in proteomics: A comprehensive review. Anal Chim Acta 2024; 1306:342513. [PMID: 38692783 DOI: 10.1016/j.aca.2024.342513] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Revised: 03/19/2024] [Accepted: 03/20/2024] [Indexed: 05/03/2024]
Abstract
Over the past decades, the proteomics field has undergone rapid growth. Progress in mass spectrometry and bioinformatics, together with separation methods, has brought many innovative approaches to the study of the molecular biology of the cell. The potential of affinity chromatography was recognized immediately after its first application in proteomics, and since that time, it has become one of the cornerstones of many proteomic protocols. Indeed, this chromatographic technique exploiting the specific binding between two molecules has been employed for numerous purposes, from selective removal of interfering (over)abundant proteins or enrichment of scarce biomarkers in complex biological samples to mapping the post-translational modifications and protein interactions with other proteins, nucleic acids or biologically active small molecules. This review presents a comprehensive survey of this versatile analytical tool in current proteomics. To navigate the reader, the haphazard space of affinity separations is classified according to the experiment's aims and the separated molecule's nature. Different types of available ligands and experimental strategies are discussed in further detail for each of the mentioned procedures.
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Affiliation(s)
- Ivo Chamrád
- Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany of the Czech Academy of Sciences, Šlechtitelů 241/27, CZ-77900, Olomouc, Holice, Czech Republic.
| | - Radim Simerský
- Department of Chemical Biology, Faculty of Science, Palacký University, Šlechtitelů 241/27, CZ-77900, Olomouc, Holice, Czech Republic
| | - René Lenobel
- Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany of the Czech Academy of Sciences, Šlechtitelů 241/27, CZ-77900, Olomouc, Holice, Czech Republic
| | - Ondřej Novák
- Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany of the Czech Academy of Sciences, Šlechtitelů 241/27, CZ-77900, Olomouc, Holice, Czech Republic
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10
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Rupasinghe M, Bersaglieri C, Leslie Pedrioli DM, Pedrioli PG, Panatta M, Hottiger MO, Cinelli P, Santoro R. PRAMEL7 and CUL2 decrease NuRD stability to establish ground-state pluripotency. EMBO Rep 2024; 25:1453-1468. [PMID: 38332149 PMCID: PMC10933316 DOI: 10.1038/s44319-024-00083-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Revised: 01/05/2024] [Accepted: 01/10/2024] [Indexed: 02/10/2024] Open
Abstract
Pluripotency is established in E4.5 preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of pluripotency, however, their gene expression signature only partially resembles that of developmental ground-state. Induced PRAMEL7 expression, a protein highly expressed in the ICM but lowly expressed in ESCs, reprograms developmentally advanced ESC+serum into ground-state pluripotency by inducing a gene expression signature close to developmental ground-state. However, how PRAMEL7 reprograms gene expression remains elusive. Here we show that PRAMEL7 associates with Cullin2 (CUL2) and this interaction is required to establish ground-state gene expression. PRAMEL7 recruits CUL2 to chromatin and targets regulators of repressive chromatin, including the NuRD complex, for proteasomal degradation. PRAMEL7 antagonizes NuRD-mediated repression of genes implicated in pluripotency by decreasing NuRD stability and promoter association in a CUL2-dependent manner. Our data link proteasome degradation pathways to ground-state gene expression, offering insights to generate in vitro models to reproduce the in vivo ground-state pluripotency.
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Affiliation(s)
- Meneka Rupasinghe
- Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, 8057, Zurich, Switzerland
- Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, 8057, Zurich, Switzerland
| | - Cristiana Bersaglieri
- Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, 8057, Zurich, Switzerland
| | - Deena M Leslie Pedrioli
- Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, 8057, Zurich, Switzerland
| | - Patrick Ga Pedrioli
- Department of Health Sciences and Technology, ETH Zurich, 8093, Zurich, Switzerland
- Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland
| | - Martina Panatta
- Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, 8057, Zurich, Switzerland
- RNA Biology Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland
| | - Michael O Hottiger
- Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, 8057, Zurich, Switzerland
| | - Paolo Cinelli
- Department of Trauma Surgery, University Hospital Zurich, University of Zurich, Rämistrasse 100, 8091, Zurich, Switzerland
| | - Raffaella Santoro
- Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, 8057, Zurich, Switzerland.
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11
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G Jagadeeshaprasad M, Zeng J, Zheng N. LC-MS bioanalysis of protein biomarkers and protein therapeutics in formalin-fixed paraffin-embedded tissue specimens. Bioanalysis 2024; 16:245-258. [PMID: 38226835 DOI: 10.4155/bio-2023-0210] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2024] Open
Abstract
Formalin-fixed paraffin-embedded (FFPE) is a form of preservation and preparation for biopsy specimens. FFPE tissue specimens are readily available as part of oncology studies because they are often collected for disease diagnosis or confirmation. FFPE tissue specimens could be extremely useful for retrospective studies on protein biomarkers because the samples preserved in FFPE blocks could be stable for decades. However, LC-MS bioanalysis of FFPE tissues poses significant challenges. In this Perspective, we review the benefits and recent developments in LC-MS approach for targeted protein biomarker and protein therapeutic analysis using FFPE tissues and their clinical and translational applications. We believe that LC-MS bioanalysis of protein biomarkers in FFPE tissue specimens represents a great potential for its clinical applications.
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Affiliation(s)
| | - Jianing Zeng
- Department of Protein Sciences & Mass Spectrometry, Translational Medicine, Bristol Myers Squibb, Princeton, NJ 08543, USA
| | - Naiyu Zheng
- Department of Protein Sciences & Mass Spectrometry, Translational Medicine, Bristol Myers Squibb, Princeton, NJ 08543, USA
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12
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Darville LNF, Lockhart JH, Putty Reddy S, Fang B, Izumi V, Boyle TA, Haura EB, Flores ER, Koomen JM. A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues. Methods Mol Biol 2024; 2823:193-223. [PMID: 39052222 PMCID: PMC11648944 DOI: 10.1007/978-1-0716-3922-1_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/27/2024]
Abstract
Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).
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Affiliation(s)
| | | | | | - Bin Fang
- H. Lee Moffitt Cancer Center, Tampa, FL, USA
| | | | | | | | | | - John M Koomen
- H. Lee Moffitt Cancer Center, Tampa, FL, USA.
- Molecular Oncology/Pathology, Moffitt Cancer Center, Tampa, FL, USA.
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13
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Güldenpfennig A, Hopp AK, Muskalla L, Manetsch P, Raith F, Hellweg L, Dördelmann C, Leslie Pedrioli D, Johnsson K, Superti-Furga G, Hottiger M. Absence of mitochondrial SLC25A51 enhances PARP1-dependent DNA repair by increasing nuclear NAD+ levels. Nucleic Acids Res 2023; 51:9248-9265. [PMID: 37587695 PMCID: PMC10516648 DOI: 10.1093/nar/gkad659] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Accepted: 07/31/2023] [Indexed: 08/18/2023] Open
Abstract
Though the effect of the recently identified mitochondrial NAD+ transporter SLC25A51 on glucose metabolism has been described, its contribution to other NAD+-dependent processes throughout the cell such as ADP-ribosylation remains elusive. Here, we report that absence of SLC25A51 leads to increased NAD+ concentration not only in the cytoplasm and but also in the nucleus. The increase is not associated with upregulation of the salvage pathway, implying an accumulation of constitutively synthesized NAD+ in the cytoplasm and nucleus. This results in an increase of PARP1-mediated nuclear ADP-ribosylation, as well as faster repair of DNA lesions induced by different single-strand DNA damaging agents. Lastly, absence of SLC25A51 reduces both MMS/Olaparib induced PARP1 chromatin retention and the sensitivity of different breast cancer cells to PARP1 inhibition. Together these results provide evidence that SLC25A51 might be a novel target to improve PARP1 inhibitor based therapies by changing subcellular NAD+ redistribution.
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Affiliation(s)
- Anka Güldenpfennig
- Department of Molecular Mechanisms of Disease (DMMD), University of Zurich, 8057 Zurich, Switzerland
- Life Science Zurich Graduate School, Molecular Life Science Ph.D. Program, University of Zurich, 8057 Zurich, Switzerland
| | - Ann-Katrin Hopp
- Department of Molecular Mechanisms of Disease (DMMD), University of Zurich, 8057 Zurich, Switzerland
- Research Center for Molecular Medicine (CeMM) of the Austrian Academy of Science, 1090 Vienna, Austria
| | - Lukas Muskalla
- Department of Molecular Mechanisms of Disease (DMMD), University of Zurich, 8057 Zurich, Switzerland
- Life Science Zurich Graduate School, Cancer Biology Ph.D. Program, University of Zurich, 8057 Zurich, Switzerland
| | - Patrick Manetsch
- Department of Molecular Mechanisms of Disease (DMMD), University of Zurich, 8057 Zurich, Switzerland
- Life Science Zurich Graduate School, Molecular Life Science Ph.D. Program, University of Zurich, 8057 Zurich, Switzerland
| | - Fabio Raith
- Department of Chemical Biology, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany
| | - Lars Hellweg
- Department of Chemical Biology, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany
| | - Cyril Dördelmann
- Life Science Zurich Graduate School, Cancer Biology Ph.D. Program, University of Zurich, 8057 Zurich, Switzerland
- Institute of Molecular Cancer Research (IMCR), University of Zurich, 8057 Zurich, Switzerland
| | - Deena M Leslie Pedrioli
- Department of Molecular Mechanisms of Disease (DMMD), University of Zurich, 8057 Zurich, Switzerland
| | - Kai Johnsson
- Department of Chemical Biology, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany
- Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL) Lausanne, 1015 Lausanne, Switzerland
| | - Giulio Superti-Furga
- Research Center for Molecular Medicine (CeMM) of the Austrian Academy of Science, 1090 Vienna, Austria
- Center for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria
| | - Michael O Hottiger
- Department of Molecular Mechanisms of Disease (DMMD), University of Zurich, 8057 Zurich, Switzerland
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14
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Carnielli CM, Melo de Lima Morais T, Malta de Sá Patroni F, Prado Ribeiro AC, Brandão TB, Sobroza E, Matos LL, Kowalski LP, Paes Leme AF, Kawahara R, Thaysen-Andersen M. Comprehensive glycoprofiling of oral tumours associates N-glycosylation with lymph node metastasis and patient survival. Mol Cell Proteomics 2023:100586. [PMID: 37268159 PMCID: PMC10336694 DOI: 10.1016/j.mcpro.2023.100586] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Revised: 05/08/2023] [Accepted: 05/30/2023] [Indexed: 06/04/2023] Open
Abstract
While altered protein glycosylation is regarded a trait of oral squamous cell carcinoma (OSCC), the heterogeneous and dynamic glycoproteome of tumour tissues from OSCC patients remain unmapped. To this end, we here employ an integrated multi-omics approach comprising unbiased and quantitative glycomics and glycoproteomics applied to a cohort of resected primary tumour tissues from OSCC patients with (n = 19) and without (n = 12) lymph node metastasis. While all tumour tissues displayed relatively uniform N-glycome profiles suggesting overall stable global N-glycosylation during disease progression, altered expression of six sialylated N-glycans was found to correlate with lymph node metastasis. Notably, glycoproteomics and advanced statistical analyses uncovered altered site-specific N-glycosylation revealing previously unknown associations with several clinicopathological features. Importantly, the glycomics and glycoproteomics data unveiled that comparatively high abundance of two core-fucosylated and sialylated N-glycans (Glycan 40a and Glycan 46a) and one N-glycopeptide from fibronectin were associated with low patient survival, while a relatively low abundance of N-glycopeptides from both afamin and CD59 were also associated with poor survival. This study provides novel insight into the complex OSCC tissue N-glycoproteome forming an important resource to further explore the underpinning disease mechanisms and uncover new prognostic glyco-markers for OSCC.
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Affiliation(s)
- Carolina Moretto Carnielli
- Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, 13083-970 SP, Brazil
| | | | | | - Ana Carolina Prado Ribeiro
- Serviço de Odontologia Oncológica, Instituto do Câncer do Estado de São Paulo, ICESP-FMUSP, São Paulo, 01246-000 SP, Brazil; Universidade Brasil, Fernandópolis, 15600-000 SP, Brazil
| | - Thaís Bianca Brandão
- Serviço de Odontologia Oncológica, Instituto do Câncer do Estado de São Paulo, ICESP-FMUSP, São Paulo, 01246-000 SP, Brazil
| | - Evandro Sobroza
- Serviço de Odontologia Oncológica, Instituto do Câncer do Estado de São Paulo, ICESP-FMUSP, São Paulo, 01246-000 SP, Brazil
| | - Leandro Luongo Matos
- Serviço de Cirurgia de Cabeça e Pescoço, Instituto do Câncer do Estado de São Paulo, ICESP-FMUSP, São Paulo, 01246-000 SP, Brazil
| | - Luiz Paulo Kowalski
- Departamento de Cirurgia de Cabeça e Pescoço e Otorrinolaringologia, A.C. Camargo Cancer Center, São Paulo, SP, 01509-900, Brazil; Departamento de Cirurgia de Cabeça e Pescoço, Faculdade de Medicina, Universidade de São Paulo - USP, São Paulo, SP, 01246-903, Brazil
| | - Adriana Franco Paes Leme
- Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, 13083-970 SP, Brazil.
| | - Rebeca Kawahara
- School of Natural Sciences, Macquarie University, Sydney, NSW-2109, Australia; Institute for Glyco-core Research (iGCORE), Nagoya University, Nagoya, 464-8601, Japan.
| | - Morten Thaysen-Andersen
- School of Natural Sciences, Macquarie University, Sydney, NSW-2109, Australia; Institute for Glyco-core Research (iGCORE), Nagoya University, Nagoya, 464-8601, Japan.
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15
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Long L, Wang W, Liu Z, Lin Y, Wang J, Lin Q, Ding S. Insights into the capability of the lignocellulolytic enzymes of Penicillium parvum 4-14 to saccharify corn bran after alkaline hydrogen peroxide pretreatment. BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS 2023; 16:79. [PMID: 37170321 PMCID: PMC10176746 DOI: 10.1186/s13068-023-02319-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/15/2023] [Accepted: 04/10/2023] [Indexed: 05/13/2023]
Abstract
BACKGROUND Corn bran is a major agro-industrial byproduct from corn starch processing. It contains abundant arabinoxylan that can be converted into value-added chemicals via biotechnology. Corn bran arabinoxylan (CBAX) is one of the most recalcitrant xylans for enzymatic degradation due to its particular heterogeneous nature. The present study aimed to investigate the capability of the filamentous fungus Penicillium parvum 4-14 to enzymatically saccharify CBAX and reveal the fungal carbohydrate-active enzyme (CAZyme) repertoire by genome sequencing and secretome analysis. RESULTS CBAX1 and CBAX2 with different branching degrees, together with corn bran residue (CBR) were generated from corn bran after alkaline hydrogen peroxide (AHP) pretreatment and graded ethanol precipitation. The protein blends E_CBAX1, E_CBAX2, and E_CBR were produced by the fungus grown on CBAX1, CBAX2, or CBR, respectively. Under the optimal conditions, E_CBAX1 released more than 80% xylose and arabinose from CBAX1 and CBAX2. Almost complete saccharification of the arabinoxylans was achieved by combining E_CBAX1 and a commercial enzyme cocktail Cellic®CTec3. Approximately 89% glucose, 64% xylose, and 64% arabinose were liberated from CBR by E_CBR. The combination of E_CBR with Cellic®CTec3 enhanced the saccharification of CBR, with conversion ratios of 97% for glucose, 81% for xylose, and 76% for arabinose. A total of 376 CAZymes including plentiful lignocellulolytic enzymes were predicted in P. parvum based on the fungal genomic sequence (25.8 Mb). Proteomic analysis indicated that the expression of CAZymes in P. parvum varied between CBAX1 and CBR, and the fungus produced complete cellulases, numerous hemicellulases, as well as high levels of glycosidases under the culture conditions. CONCLUSIONS This investigation disclosed the CAZyme repertoire of P. parvum at the genomic and proteomic levels, and elaborated on the promising potential of fungal lignocellulolytic enzymes upon saccharification of corn bran biomass after AHP pretreatment.
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Affiliation(s)
- Liangkun Long
- Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China
- Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, Nanjing Forestry University, Nanjing, 210037, China
| | - Wei Wang
- Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China
| | - Zhen Liu
- Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China
| | - Yuanxin Lin
- Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China
| | - Jing Wang
- Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China
| | - Qunying Lin
- Nanjing Institute for the Comprehensive Utilization of Wild Plants, Nanjing, 211111, China
| | - Shaojun Ding
- Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China.
- Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, Nanjing Forestry University, Nanjing, 210037, China.
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16
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Barnabas G, Goebeler V, Tsui J, Bush JW, Lange PF. ASAP─Automated Sonication-Free Acid-Assisted Proteomes─from Cells and FFPE Tissues. Anal Chem 2023; 95:3291-3299. [PMID: 36724070 PMCID: PMC9933881 DOI: 10.1021/acs.analchem.2c04264] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2022] [Accepted: 01/17/2023] [Indexed: 02/02/2023]
Abstract
Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens.
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Affiliation(s)
- Georgina
D. Barnabas
- Department
of Pathology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
- Michael
Cuccione Childhood Cancer Research Program, BC Children’s Hospital and Research Institute, Vancouver, British Columbia V5Z 4H4, Canada
| | - Verena Goebeler
- Department
of Pediatrics, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
- Michael
Cuccione Childhood Cancer Research Program, BC Children’s Hospital and Research Institute, Vancouver, British Columbia V5Z 4H4, Canada
| | - Janice Tsui
- Department
of Pathology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
- Michael
Cuccione Childhood Cancer Research Program, BC Children’s Hospital and Research Institute, Vancouver, British Columbia V5Z 4H4, Canada
| | - Jonathan W. Bush
- Department
of Pathology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
| | - Philipp F. Lange
- Department
of Pathology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
- Michael
Cuccione Childhood Cancer Research Program, BC Children’s Hospital and Research Institute, Vancouver, British Columbia V5Z 4H4, Canada
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17
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Tang R, Zhu D, Luo Y, He D, Zhang H, El-Naggar A, Palansooriya KN, Chen K, Yan Y, Lu X, Ying M, Sun T, Cao Y, Diao Z, Zhang Y, Lian Y, Chang SX, Cai Y. Nanoplastics induce molecular toxicity in earthworm: Integrated multi-omics, morphological, and intestinal microorganism analyses. JOURNAL OF HAZARDOUS MATERIALS 2023; 442:130034. [PMID: 36206716 DOI: 10.1016/j.jhazmat.2022.130034] [Citation(s) in RCA: 42] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 09/02/2022] [Accepted: 09/18/2022] [Indexed: 06/16/2023]
Abstract
The toxicity of nanoplastics (NPs) at relatively low concentrations to soil fauna at different organismal levels is poorly understood. We investigated the responses of earthworm (Eisenia fetida) to polystyrene NPs (90-110 nm) contaminated soil at a relatively low concentration (0.02 % w:w) based on multi-omics, morphological, and intestinal microorganism analyses. Results showed that NPs accumulated in earthworms' intestinal tissues. The NPs damaged earthworms' digestive and immune systems based on injuries of the intestinal epithelium and chloragogenous tissues (tissue level) and increased the number of changed genes in the digestive and immune systems (transcriptome level). The NPs reduced gut microorganisms' diversity (Shannon index) and species richness (Chao 1 index). Proteomic, transcriptome, and histopathological analyses showed that earthworms suffered from oxidative and inflammatory stresses. Moreover, NPs influenced the osmoregulatory metabolism of earthworms as NPs damaged intestinal epithelium (tissue level), increased aldosterone-regulated sodium reabsorption (transcriptome level), inositol phosphate metabolism (proteomic level) and 2-hexyl-5-ethyl-furan-3-sulfonic acid, and decreased betaine and myo-inositol concentrations (metabolic level). Transcriptional-metabolic and transcriptional-proteomic analyses revealed that NPs disrupted earthworm carbohydrate and arachidonic acid metabolisms. Our multi-level investigation indicates that NPs at a relatively low concentration induced toxicity to earthworms and suggests that NPs pollution has significant environmental toxicity risks for soil fauna.
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Affiliation(s)
- Ronggui Tang
- State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, China
| | - Dong Zhu
- State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
| | - Yongming Luo
- CAS Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China
| | - Defu He
- School of Ecological and Environmental Sciences, East China Normal University, Shanghai 200241, China
| | - Haibo Zhang
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Ali El-Naggar
- State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, China; Department of Soil Sciences, Faculty of Agriculture, Ain Shams University, Cairo 11241, Egypt
| | - Kumuduni Niroshika Palansooriya
- State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, China; College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Keyi Chen
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Yan Yan
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Xinghang Lu
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Minshen Ying
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Tao Sun
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Yuntao Cao
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Zhihan Diao
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Yuxin Zhang
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Yichen Lian
- College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China
| | - Scott X Chang
- State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, China; Department of Renewable Resources, University of Alberta, Edmonton T6G2E3, Canada.
| | - Yanjiang Cai
- State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, China
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18
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Panjaitan FCA, Chen TY, Ku HH, Chang YW. In Silico and In Vitro Analyses of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Activities of Enzymatic Protein Hydrolysates from Taiwan Mackerel ( Scomber australasicus) Steaming Juice. Foods 2022; 11:foods11121785. [PMID: 35741983 PMCID: PMC9222390 DOI: 10.3390/foods11121785] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Revised: 06/13/2022] [Accepted: 06/15/2022] [Indexed: 01/27/2023] Open
Abstract
Mackerel (Scomber australasicus) steaming juice (MSJ) can be a good source of proteins. However, it is often treated as food waste during the canning process. The objective of this study was to investigate the Angiotensin-I converting enzyme (ACE-I) inhibitory and antioxidant activities from MSJ hydrolysates using in silico and in vitro approaches. Proteins extracted from MSJ were identified by proteomic techniques, followed by sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in-gel digestion, tandem mass spectrometry and on-line Mascot database analysis. Myosin heavy chain (fast skeletal muscle), actin, myosin light chain 1 (skeletal muscle isoform), collagen alpha-2(I) chain, tropomyosin alpha-1 chain, beta-enolase, fructose-bisphosphate aldolase A and glyceraldehyde-3- phosphate dehydrogenase were identified and further analyzed using BIOPEP-UWM database. In silico results indicated that MSJ proteins had potential bioactive peptides of antioxidant and ACE-I inhibitory activities. MSJ was then hydrolyzed using six proteases (papain, pepsin, proteinase k, alcalase, bromelain, thermolysin). In particular, pepsin hydrolysates (5 mg/mL) showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (61.54%) among others. Alcalase hydrolysates (5 mg/mL) exhibited the highest metal chelating activity (89.76%) and proteinase K hydrolysates (5 mg/mL) indicated the highest reducing power activity (1.52 abs). Moreover, pepsin hydrolysates (0.1 mg/mL) possessed the highest ACE inhibitory activity (86.15%). Current findings suggest that MSJ hydrolysates can be a potential material to produce ACE-I inhibitory and antioxidant peptides as nutraceutical or pharmaceutical ingredients/products with added values.
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Affiliation(s)
- Fenny Crista A. Panjaitan
- Marine Products Processing Study Program, Marine and Fisheries Polytechnic of Jembrana, Bali 82218, Indonesia;
| | - Ting-Yi Chen
- Department of Food Science, National Taiwan Ocean University, Keelung City 20224, Taiwan;
| | - Hao-Hsiang Ku
- Institute of Food Safety and Risk Management, National Taiwan Ocean University, Keelung City 20224, Taiwan;
| | - Yu-Wei Chang
- Department of Food Science, National Taiwan Ocean University, Keelung City 20224, Taiwan;
- Correspondence: ; Tel.: +886-2-2462-2192 (ext. 5152)
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19
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Mund A, Brunner AD, Mann M. Unbiased spatial proteomics with single-cell resolution in tissues. Mol Cell 2022; 82:2335-2349. [PMID: 35714588 DOI: 10.1016/j.molcel.2022.05.022] [Citation(s) in RCA: 133] [Impact Index Per Article: 44.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2022] [Revised: 05/05/2022] [Accepted: 05/18/2022] [Indexed: 12/19/2022]
Abstract
Mass spectrometry (MS)-based proteomics has become a powerful technology to quantify the entire complement of proteins in cells or tissues. Here, we review challenges and recent advances in the LC-MS-based analysis of minute protein amounts, down to the level of single cells. Application of this technology revealed that single-cell transcriptomes are dominated by stochastic noise due to the very low number of transcripts per cell, whereas the single-cell proteome appears to be complete. The spatial organization of cells in tissues can be studied by emerging technologies, including multiplexed imaging and spatial transcriptomics, which can now be combined with ultra-sensitive proteomics. Combined with high-content imaging, artificial intelligence and single-cell laser microdissection, MS-based proteomics provides an unbiased molecular readout close to the functional level. Potential applications range from basic biological questions to precision medicine.
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Affiliation(s)
- Andreas Mund
- Proteomics Program, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Andreas-David Brunner
- Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany; Boehringer Ingelheim Pharma GmbH & Co. KG, Drug Discovery Sciences, Birkendorfer Str. 65, D-88397, Biberach Riss, Germany
| | - Matthias Mann
- Proteomics Program, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3B, 2200 Copenhagen, Denmark; Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
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20
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Bennett APS, de la Torre-Escudero E, Dermott SSE, Threadgold LT, Hanna REB, Robinson MW. Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism. Int J Mol Sci 2022; 23:ijms23105525. [PMID: 35628335 PMCID: PMC9143473 DOI: 10.3390/ijms23105525] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2022] [Revised: 05/09/2022] [Accepted: 05/12/2022] [Indexed: 02/01/2023] Open
Abstract
The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host’s blood. Despite their importance in the host–parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze–thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite.
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Affiliation(s)
- Adam P. S. Bennett
- School of Biological Sciences, The Queen’s University of Belfast, Belfast BT9 5DL, UK; (A.P.S.B.); (E.d.l.T.-E.)
| | - Eduardo de la Torre-Escudero
- School of Biological Sciences, The Queen’s University of Belfast, Belfast BT9 5DL, UK; (A.P.S.B.); (E.d.l.T.-E.)
| | - Susan S. E. Dermott
- School of Biological Sciences, The Queen’s University of Belfast, Belfast BT9 5DL, UK; (A.P.S.B.); (E.d.l.T.-E.)
| | - Lawrence T. Threadgold
- School of Biological Sciences, The Queen’s University of Belfast, Belfast BT9 5DL, UK; (A.P.S.B.); (E.d.l.T.-E.)
| | - Robert E. B. Hanna
- Veterinary Sciences Division, Agri-Food and Biosciences Institute (AFBI), Stormont, Belfast BT4 3SD, UK;
| | - Mark W. Robinson
- School of Biological Sciences, The Queen’s University of Belfast, Belfast BT9 5DL, UK; (A.P.S.B.); (E.d.l.T.-E.)
- Correspondence: ; Tel.: +44-(0)28-9097-2120
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21
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Vessby J, Wisniewski JR, Lindskog C, Eriksson N, Gabrysch K, Zettl K, Wanders A, Carlson M, Rorsman F, Åberg M. AGPAT1 as a Novel Colonic Biomarker for Discriminating Between Ulcerative Colitis With and Without Primary Sclerosing Cholangitis. Clin Transl Gastroenterol 2022; 13:e00486. [PMID: 35363634 PMCID: PMC9132532 DOI: 10.14309/ctg.0000000000000486] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 03/15/2022] [Indexed: 11/17/2022] Open
Abstract
INTRODUCTION Ulcerative colitis (UC) associated with primary sclerosing cholangitis (PSC-UC) is considered a unique inflammatory bowel disease (IBD) entity. PSC diagnosis in an IBD individual entails a significantly higher risk of gastrointestinal cancer; however, biomarkers for identifying patients with UC at risk for PSC are lacking. We, therefore, performed a thorough PSC-UC biomarker study, starting from archived colonic tissue. METHODS Proteins were extracted out of formalin-fixed paraffin-embedded proximal colon samples from PSC-UC (n = 9), UC (n = 7), and healthy controls (n = 7). Patients with IBD were in clinical and histological remission, and all patients with UC had a history of pancolitis. Samples were processed by the multienzyme digestion FASP and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Candidate proteins were replicated in an independent cohort (n: PSC-UC = 16 and UC = 21) and further validated by immunohistochemistry. RESULTS In the discovery step, 7,279 unique proteins were detected. The top 5 most differentiating proteins (PSC-UC vs UC) based on linear regression analysis were selected for replication. Of these, 1-acetylglycerol-3-phosphate O-acyltransferase 1 (AGPAT1) was verified as higher in PSC-UC than UC (P = 0.009) in the replication cohort. A difference on the group level was also confirmed by immunohistochemistry, showing more intense AGPAT1 staining in patients with PSC-UC compared with UC. DISCUSSION We present AGPAT1 as a potential colonic biomarker for differentiating PSC-UC from UC. Our findings have possible implication for future PSC-IBD diagnostics and surveillance.
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Affiliation(s)
- Johan Vessby
- Department of Medical Sciences, Gastroenterology Research Group, Uppsala University, Uppsala, Sweden
| | - Jacek R. Wisniewski
- Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany;
| | - Cecilia Lindskog
- Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden;
| | - Niclas Eriksson
- Uppsala Clinical Research Center, Uppsala University, Uppsala, Sweden;
| | - Katja Gabrysch
- Uppsala Clinical Research Center, Uppsala University, Uppsala, Sweden;
| | - Katharina Zettl
- Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany;
| | - Alkwin Wanders
- Department of Pathology, Aalborg University Hospital, Aalborg, Denmark;
| | - Marie Carlson
- Department of Medical Sciences, Gastroenterology Research Group, Uppsala University, Uppsala, Sweden
| | - Fredrik Rorsman
- Department of Medical Sciences, Gastroenterology Research Group, Uppsala University, Uppsala, Sweden
| | - Mikael Åberg
- Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
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22
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The emerging role of mass spectrometry-based proteomics in drug discovery. Nat Rev Drug Discov 2022; 21:637-654. [PMID: 35351998 DOI: 10.1038/s41573-022-00409-3] [Citation(s) in RCA: 167] [Impact Index Per Article: 55.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/19/2022] [Indexed: 12/14/2022]
Abstract
Proteins are the main targets of most drugs; however, system-wide methods to monitor protein activity and function are still underused in drug discovery. Novel biochemical approaches, in combination with recent developments in mass spectrometry-based proteomics instrumentation and data analysis pipelines, have now enabled the dissection of disease phenotypes and their modulation by bioactive molecules at unprecedented resolution and dimensionality. In this Review, we describe proteomics and chemoproteomics approaches for target identification and validation, as well as for identification of safety hazards. We discuss innovative strategies in early-stage drug discovery in which proteomics approaches generate unique insights, such as targeted protein degradation and the use of reactive fragments, and provide guidance for experimental strategies crucial for success.
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23
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Weke K, Kote S, Faktor J, Al Shboul S, Uwugiaren N, Brennan PM, Goodlett DR, Hupp TR, Dapic I. DIA-MS proteome analysis of formalin-fixed paraffin-embedded glioblastoma tissues. Anal Chim Acta 2022; 1204:339695. [DOI: 10.1016/j.aca.2022.339695] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2021] [Revised: 03/04/2022] [Accepted: 03/05/2022] [Indexed: 12/11/2022]
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24
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Paulsen B, Velasco S, Kedaigle AJ, Pigoni M, Quadrato G, Deo AJ, Adiconis X, Uzquiano A, Sartore R, Yang SM, Simmons SK, Symvoulidis P, Kim K, Tsafou K, Podury A, Abbate C, Tucewicz A, Smith SN, Albanese A, Barrett L, Sanjana NE, Shi X, Chung K, Lage K, Boyden ES, Regev A, Levin JZ, Arlotta P. Autism genes converge on asynchronous development of shared neuron classes. Nature 2022; 602:268-273. [PMID: 35110736 PMCID: PMC8852827 DOI: 10.1038/s41586-021-04358-6] [Citation(s) in RCA: 230] [Impact Index Per Article: 76.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2020] [Accepted: 12/14/2021] [Indexed: 02/08/2023]
Abstract
Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.
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Affiliation(s)
- Bruna Paulsen
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Silvia Velasco
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
- Murdoch Children's Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia.
| | - Amanda J Kedaigle
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Martina Pigoni
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Giorgia Quadrato
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Anthony J Deo
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Department of Psychiatry, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
- Department of Psychiatry, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ, USA
- Rutgers University Behavioral Health Care, Piscataway, NJ, USA
| | - Xian Adiconis
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Ana Uzquiano
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Rafaela Sartore
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Sung Min Yang
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Sean K Simmons
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Panagiotis Symvoulidis
- MIT Center for Neurobiological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
| | - Kwanho Kim
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Kalliopi Tsafou
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Archana Podury
- MIT Center for Neurobiological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- Harvard-MIT Health Sciences & Technology Program (HST), Harvard Medical School, Boston, MA, USA
| | - Catherine Abbate
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Ashley Tucewicz
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Samantha N Smith
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Alexandre Albanese
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
| | - Lindy Barrett
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Neville E Sanjana
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- New York Genome Center, New York, NY, USA
- Department of Biology, New York University, New York, NY, USA
| | - Xi Shi
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Kwanghun Chung
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- Picower Institute for Learning and Memory, Departments of Chemical Engineering and Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- New York Genome Center, New York, NY, USA
| | - Kasper Lage
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Department of Surgery and Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA
| | - Edward S Boyden
- MIT Center for Neurobiological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- Harvard-MIT Health Sciences & Technology Program (HST), Harvard Medical School, Boston, MA, USA
- McGovern Institute for Brain Research, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- Howard Hughes Medical Institute, MIT, Cambridge, MA, USA
- Department of Brain of Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
| | - Aviv Regev
- Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
- Genentech, South San Francisco, CA, USA
| | - Joshua Z Levin
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Paola Arlotta
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
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25
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Barbarics B, Eildermann K, Kaderali L, Cyganek L, Plessmann U, Bodemeyer J, Paul T, Ströbel P, Urlaub H, Tirilomis T, Lenz C, Bohnenberger H. Proteomic mapping of atrial and ventricular heart tissue in patients with aortic valve stenosis. Sci Rep 2021; 11:24389. [PMID: 34937869 PMCID: PMC8695579 DOI: 10.1038/s41598-021-03907-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Accepted: 12/06/2021] [Indexed: 11/20/2022] Open
Abstract
Aortic valve stenosis (AVS) is one of the most common valve diseases in the world. However, detailed biological understanding of the myocardial changes in AVS hearts on the proteome level is still lacking. Proteomic studies using high-resolution mass spectrometry of formalin-fixed and paraffin-embedded (FFPE) human myocardial tissue of AVS-patients are very rare due to methodical issues. To overcome these issues this study used high resolution mass spectrometry in combination with a stem cell-derived cardiac specific protein quantification-standard to profile the proteomes of 17 atrial and 29 left ventricular myocardial FFPE human myocardial tissue samples from AVS-patients. In our proteomic analysis we quantified a median of 1980 (range 1495–2281) proteins in every single sample and identified significant upregulation of 239 proteins in atrial and 54 proteins in ventricular myocardium. We compared the proteins with published data. Well studied proteins reflect disease-related changes in AVS, such as cardiac hypertrophy, development of fibrosis, impairment of mitochondria and downregulated blood supply. In summary, we provide both a workflow for quantitative proteomics of human FFPE heart tissue and a comprehensive proteomic resource for AVS induced changes in the human myocardium.
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26
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Yin B, Caggiano LR, Li RC, McGowan E, Holmes JW, Ewald SE. Automated Spatially Targeted Optical Microproteomics Investigates Inflammatory Lesions In Situ. J Proteome Res 2021; 20:4543-4552. [PMID: 34436902 PMCID: PMC8969901 DOI: 10.1021/acs.jproteome.1c00505] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
![]()
Tissue
microenvironment properties like blood flow, extracellular
matrix, or proximity to immune-infiltrate are important regulators
of cell biology. However, methods to study regional protein expression
in the native tissue environment are limited. To address this need,
we developed a novel approach to visualize, purify, and measure proteins in situ using automated spatially targeted optical microproteomics
(AutoSTOMP). Here, we report custom codes to specify regions of heterogeneity
in a tissue section and UV-biotinylate proteins within those regions.
We have developed liquid chromatography–mass spectrometry (LC–MS)/MS-compatible
biochemistry to purify those proteins and label-free quantification
methodology to determine protein enrichment in target cell types or
structures relative to nontarget regions in the same sample. These
tools were applied to (a) identify inflammatory proteins expressed
by CD68+ macrophages in rat cardiac infarcts and (b) characterize
inflammatory proteins enriched in IgG4+ lesions in human
esophageal tissues. These data indicate that AutoSTOMP is a flexible
approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current
tools and sample availability are limited.
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Affiliation(s)
- Bocheng Yin
- Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, Virginia 22903, United States
| | - Laura R Caggiano
- Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, Virginia 22903, United States
| | - Rung-Chi Li
- Division of Allergy and Clinical Immunology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22903, United States.,Department of Allergy and Immunology, Northern Light Health, Bangor, Maine 04401, United States
| | - Emily McGowan
- Division of Allergy and Clinical Immunology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22903, United States
| | - Jeffrey W Holmes
- Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, Virginia 22903, United States.,School of Engineering, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States
| | - Sarah E Ewald
- Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, Virginia 22903, United States
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27
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Glass JB, Ranjan P, Kretz CB, Nunn BL, Johnson AM, Xu M, McManus J, Stewart FJ. Microbial metabolism and adaptations in Atribacteria-dominated methane hydrate sediments. Environ Microbiol 2021; 23:4646-4660. [PMID: 34190392 DOI: 10.1111/1462-2920.15656] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2019] [Accepted: 06/28/2021] [Indexed: 12/12/2022]
Abstract
Gas hydrates harbour gigatons of natural gas, yet their microbiomes remain understudied. We bioprospected 16S rRNA amplicons, metagenomes, and metaproteomes from methane hydrate-bearing sediments under Hydrate Ridge (offshore Oregon, USA, ODP Site 1244, 2-69 mbsf) for novel microbial metabolic and biosynthetic potential. Atribacteria sequences generally increased in relative sequence abundance with increasing sediment depth. Most Atribacteria ASVs belonged to JS-1-Genus 1 and clustered with other sequences from gas hydrate-bearing sediments. We recovered 21 metagenome-assembled genomic bins spanning three geochemical zones in the sediment core: the sulfate-methane transition zone, the metal (iron/manganese) reduction zone, and the gas hydrate stability zone. We found evidence for bacterial fermentation as a source of acetate for aceticlastic methanogenesis and as a driver of iron reduction in the metal reduction zone. In multiple zones, we identified a Ni-Fe hydrogenase-Na+ /H+ antiporter supercomplex (Hun) in Atribacteria and Firmicutes bins and in other deep subsurface bacteria and cultured hyperthermophiles from the Thermotogae phylum. Atribacteria expressed tripartite ATP-independent transporters downstream from a novel regulator (AtiR). Atribacteria also possessed adaptations to survive extreme conditions (e.g. high salt brines, high pressure and cold temperatures) including the ability to synthesize the osmolyte di-myo-inositol-phosphate as well as expression of K+ -stimulated pyrophosphatase and capsule proteins.
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Affiliation(s)
- Jennifer B Glass
- School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA, USA
| | - Piyush Ranjan
- School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA
| | | | - Brook L Nunn
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Abigail M Johnson
- School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA, USA
| | - Manlin Xu
- School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA, USA
| | - James McManus
- Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA
| | - Frank J Stewart
- School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA.,Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA
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28
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Friedrich C, Schallenberg S, Kirchner M, Ziehm M, Niquet S, Haji M, Beier C, Neudecker J, Klauschen F, Mertins P. Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories. Nat Commun 2021; 12:3576. [PMID: 34117251 PMCID: PMC8196151 DOI: 10.1038/s41467-021-23855-w] [Citation(s) in RCA: 53] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Accepted: 05/17/2021] [Indexed: 02/07/2023] Open
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable resource for retrospective clinical studies. Here, we evaluate the feasibility of (phospho-)proteomics on FFPE lung tissue regarding protein extraction, quantification, pre-analytics, and sample size. After comparing protein extraction protocols, we use the best-performing protocol for the acquisition of deep (phospho-)proteomes from lung squamous cell and adenocarcinoma with >8,000 quantified proteins and >14,000 phosphosites with a tandem mass tag (TMT) approach. With a microscaled approach, we quantify 7,000 phosphosites, enabling the analysis of FFPE biopsies with limited tissue amounts. We also investigate the influence of pre-analytical variables including fixation time and heat-assisted de-crosslinking on protein extraction efficiency and proteome coverage. Our improved workflows provide quantitative information on protein abundance and phosphosite regulation for the most relevant oncogenes, tumor suppressors, and signaling pathways in lung cancer. Finally, we present general guidelines to which methods are best suited for different applications, highlighting TMT methods for comprehensive (phospho-)proteome profiling for focused clinical studies and label-free methods for large cohorts.
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Affiliation(s)
- Corinna Friedrich
- German Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany ,grid.7497.d0000 0004 0492 0584German Cancer Research Center (DKFZ), Heidelberg, Germany ,grid.7468.d0000 0001 2248 7639Institute of Pathology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany ,grid.419491.00000 0001 1014 0849Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), MDC graduate school, Berlin, Germany ,grid.7468.d0000 0001 2248 7639Humboldt Universität zu Berlin, Institute of Chemistry, Berlin, Germany
| | - Simon Schallenberg
- grid.7468.d0000 0001 2248 7639Institute of Pathology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
| | - Marieluise Kirchner
- grid.419491.00000 0001 1014 0849Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Proteomics Platform, Berlin, Germany ,grid.484013.aBerlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Matthias Ziehm
- grid.419491.00000 0001 1014 0849Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Proteomics Platform, Berlin, Germany ,grid.484013.aBerlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Sylvia Niquet
- grid.419491.00000 0001 1014 0849Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Proteomics Platform, Berlin, Germany ,grid.484013.aBerlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Mohamed Haji
- grid.419491.00000 0001 1014 0849Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Proteomics Platform, Berlin, Germany
| | - Christin Beier
- grid.419491.00000 0001 1014 0849Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Proteomics Platform, Berlin, Germany
| | - Jens Neudecker
- grid.6363.00000 0001 2218 4662Department of Surgery - Campus Charité Mitte and Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Frederick Klauschen
- German Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany ,grid.7497.d0000 0004 0492 0584German Cancer Research Center (DKFZ), Heidelberg, Germany ,grid.7468.d0000 0001 2248 7639Institute of Pathology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany ,grid.484013.aBerlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany ,grid.5252.00000 0004 1936 973XInstitute of Pathology, Ludwig-Maximilians-Universität München, Munich, Germany
| | - Philipp Mertins
- German Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany ,grid.7497.d0000 0004 0492 0584German Cancer Research Center (DKFZ), Heidelberg, Germany ,grid.419491.00000 0001 1014 0849Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Proteomics Platform, Berlin, Germany ,grid.484013.aBerlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany
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29
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Blaschke CRK, McDowell CT, Black AP, Mehta AS, Angel PM, Drake RR. Glycan Imaging Mass Spectrometry: Progress in Developing Clinical Diagnostic Assays for Tissues, Biofluids, and Cells. Clin Lab Med 2021; 41:247-266. [PMID: 34020762 PMCID: PMC8862151 DOI: 10.1016/j.cll.2021.03.005] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
N-glycan imaging mass spectrometry (IMS) can rapidly and reproducibly identify changes in disease-associated N-linked glycosylation that are linked with histopathology features in standard formalin-fixed paraffin-embedded tissue samples. It can detect multiple N-glycans simultaneously and has been used to identify specific N-glycans and carbohydrate structural motifs as possible cancer biomarkers. Recent advancements in instrumentation and sample preparation are also discussed. The tissue N-glycan IMS workflow has been adapted to new glass slide-based assays for effective and rapid analysis of clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.
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Affiliation(s)
- Calvin R K Blaschke
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, BSB 358, Charleston, SC 29425, USA
| | - Colin T McDowell
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, BSB 358, Charleston, SC 29425, USA
| | - Alyson P Black
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, BSB 358, Charleston, SC 29425, USA
| | - Anand S Mehta
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, BSB 358, Charleston, SC 29425, USA
| | - Peggi M Angel
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, BSB 358, Charleston, SC 29425, USA
| | - Richard R Drake
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, BSB 358, Charleston, SC 29425, USA.
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30
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García-Vence M, Chantada-Vazquez MDP, Sosa-Fajardo A, Agra R, Barcia de la Iglesia A, Otero-Glez A, García-González M, Cameselle-Teijeiro JM, Nuñez C, Bravo JJ, Bravo SB. Protein Extraction From FFPE Kidney Tissue Samples: A Review of the Literature and Characterization of Techniques. Front Med (Lausanne) 2021; 8:657313. [PMID: 34055835 PMCID: PMC8158658 DOI: 10.3389/fmed.2021.657313] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2021] [Accepted: 04/01/2021] [Indexed: 12/15/2022] Open
Abstract
Most tissue biopsies from patients in hospital environments are formalin-fixed and paraffin-embedded (FFPE) for long-term storage. This fixation process produces a modification in the proteins called “crosslinks”, which improves protein stability necessary for their conservation. Currently, these samples are mainly used in clinical practice for performing immunohistochemical analysis, since these modifications do not suppose a drawback for this technique; however, crosslinks difficult the protein extraction process. Accordingly, these modifications make the development of a good protein extraction protocol necessary. Due to the specific characteristics of each tissue, the same extraction buffers or deparaffinization protocols are not equally effective in all cases. Therefore, it is necessary to obtain a specific protocol for each tissue. The present work aims to establish a deparaffinization and protein extraction protocol from FFPE kidney samples to obtain protein enough of high quality for the subsequent proteomic analysis. Different deparaffination, protocols and protein extraction buffers will be tested in FFPE kidney samples. The optimized conditions will be applied in the identification by LC-MS/MS analysis of proteins extracted from 5, 10, and 15 glomeruli obtained through the microdissection of FFPE renal samples.
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Affiliation(s)
- Maria García-Vence
- Proteomic Unit, Health Research Institute of Santiago de Compostela (IDIS), University Clinical Hospital of Santiago de Compostela (CHUS), Santiago de Compostela, Spain
| | - Maria Del Pilar Chantada-Vazquez
- Proteomic Unit, Health Research Institute of Santiago de Compostela (IDIS), University Clinical Hospital of Santiago de Compostela (CHUS), Santiago de Compostela, Spain.,Research Unit, Lucus Augusti University Hospital (HULA), Servizo Galego de Saúde (SERGAS), Lugo, Spain
| | - Ana Sosa-Fajardo
- Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Vrije Universiteit, Brussels, Belgium
| | - Rebeca Agra
- Proteomic Unit, Health Research Institute of Santiago de Compostela (IDIS), University Clinical Hospital of Santiago de Compostela (CHUS), Santiago de Compostela, Spain
| | - Ana Barcia de la Iglesia
- Nephrology Laboratory, Health Research Institute of Santiago de Compostela (IDIS), University Clinical Hospital of Santiago de Compostela (CHUS), Santiago de Compostela, Spain
| | - Alfonso Otero-Glez
- Nephrology Service, University Clinical Hospital of Ourense (CHOU), Orense, Spain
| | - Miguel García-González
- Nephrology Laboratory, Health Research Institute of Santiago de Compostela (IDIS), University Clinical Hospital of Santiago de Compostela (CHUS), Santiago de Compostela, Spain
| | - José M Cameselle-Teijeiro
- Department of Pathology, Health Research Institute of Santiago de Compostela (IDIS), University Clinical Hospital of Santiago de Compostela (CHUS), Santiago de Compostela, Santiago, Spain
| | - Cristina Nuñez
- Research Unit, Lucus Augusti University Hospital (HULA), Servizo Galego de Saúde (SERGAS), Lugo, Spain
| | - Juan J Bravo
- Nephrology Service, University Clinical Hospital of Vigo (Alvaro Cunqueiro-CHUVI), Vigo, Spain
| | - Susana B Bravo
- Proteomic Unit, Health Research Institute of Santiago de Compostela (IDIS), University Clinical Hospital of Santiago de Compostela (CHUS), Santiago de Compostela, Spain
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31
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Mann M, Brasier AR. Evolution of proteomics technologies for understanding respiratory syncytial virus pathogenesis. Expert Rev Proteomics 2021; 18:379-394. [PMID: 34018899 PMCID: PMC8277732 DOI: 10.1080/14789450.2021.1931130] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Accepted: 05/14/2021] [Indexed: 10/21/2022]
Abstract
Introduction: Respiratory syncytial virus (RSV) is a major human pathogen associated with long term morbidity. RSV replication occurs primarily in the epithelium, producing a complex cellular response associated with acute inflammation and long-lived changes in pulmonary function and allergic disease. Proteomics approaches provide important insights into post-transcriptional regulatory processes including alterations in cellular complexes regulating the coordinated innate response and epigenome.Areas covered: Peer-reviewed proteomics studies of host responses to RSV infections and proteomics techniques were analyzed. Methodologies identified include 1)." bottom-up" discovery proteomics, 2). Organellar proteomics by LC-gel fractionation; 3). Dynamic changes in protein interaction networks by LC-MS; and 4). selective reaction monitoring MS. We introduce recent developments in single-cell proteomics, top-down mass spectrometry, and photo-cleavable surfactant chemistries that will have impact on understanding how RSV induces extracellular matrix (ECM) composition and airway remodeling.Expert opinion: RSV replication induces global changes in the cellular proteome, dynamic shifts in nuclear proteins, and remodeling of epigenetic regulatory complexes linked to the innate response. Pathways discovered by proteomics technologies have led to deeper mechanistic understanding of the roles of heat shock proteins, redox response, transcriptional elongation complex remodeling and ECM secretion remodeling in host responses to RSV infections and pathological sequelae.
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Affiliation(s)
- Morgan Mann
- Department of Internal Medicine, University of Wisconsin-Madison School of Medicine and Public Health (SMPH), Madison, WI, USA
| | - Allan R Brasier
- Department of Internal Medicine and Institute for Clinical and Translational Research (ICTR), University of Wisconsin-Madison, Madison, WI, USA
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32
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Proteomic comparison between different tissue preservation methods for identification of promising biomarkers of urothelial bladder cancer. Sci Rep 2021; 11:7595. [PMID: 33828141 PMCID: PMC8027873 DOI: 10.1038/s41598-021-87003-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Accepted: 03/22/2021] [Indexed: 11/08/2022] Open
Abstract
Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1-non-muscle invasive bladder cancer (NMIBC), and T2/T3-muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers.
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33
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Gomig THB, Gontarski AM, Cavalli IJ, Souza RLRD, Lucena ACR, Batista M, Machado KC, Marchini FK, Marchi FA, Lima RS, Urban CDA, Marchi RD, Cavalli LR, Ribeiro EMDSF. Integrated analysis of label-free quantitative proteomics and bioinformatics reveal insights into signaling pathways in male breast cancer. Genet Mol Biol 2021; 44:e20190410. [PMID: 33656060 PMCID: PMC7926483 DOI: 10.1590/1678-4685-gmb-2019-0410] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2020] [Accepted: 01/18/2021] [Indexed: 01/04/2023] Open
Abstract
Male breast cancer (MBC) is a rare malignancy that accounts for about 1.8% of all breast cancer cases. In contrast to the high number of the “omics” studies in breast cancer in women, only recently molecular approaches have been performed in MBC research. High-throughput proteomics based methodologies are promisor strategies to characterize the MBC proteomic signatures and their association with clinico-pathological parameters. In this study, the label-free quantification-mass spectrometry and bioinformatics approaches were applied to analyze the proteomic profiling of a MBC case using the primary breast tumor and the corresponding axillary metastatic lymph nodes and adjacent non-tumor breast tissues. The differentially expressed proteins were identified in the signaling pathways of granzyme B, sirtuins, eIF2, actin cytoskeleton, eNOS, acute phase response and calcium and were connected to the upstream regulators MYC, PI3K SMARCA4 and cancer-related chemical drugs. An additional proteomic comparative analysis was performed with a primary breast tumor of a female patient and revealed an interesting set of proteins, which were mainly involved in cancer biology. Together, our data provide a relevant data source for the MBC research that can help the therapeutic strategies for its management.
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Affiliation(s)
| | - Amanda Moletta Gontarski
- Universidade Federal do Paraná, Departamento de Genética, Programa de Pós-graduação em Genética, Curitiba, PR, Brazil
| | - Iglenir João Cavalli
- Universidade Federal do Paraná, Departamento de Genética, Programa de Pós-graduação em Genética, Curitiba, PR, Brazil
| | | | | | - Michel Batista
- Instituto Carlos Chagas, Laboratório de Genômica Funcional, Curitiba, PR, Brazil.,Fundação Oswaldo Cruz (Fiocruz), Plataforma de Espectrometria de Massas, Curitiba, PR, Brazil
| | | | - Fabricio Klerynton Marchini
- Instituto Carlos Chagas, Laboratório de Genômica Funcional, Curitiba, PR, Brazil.,Fundação Oswaldo Cruz (Fiocruz), Plataforma de Espectrometria de Massas, Curitiba, PR, Brazil
| | | | - Rubens Silveira Lima
- Hospital Nossa Senhora das Graças, Centro de Doenças da Mama, Curitiba, PR, Brazil
| | | | | | - Luciane Regina Cavalli
- Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, PR, Brazil.,Georgetown University, Lombardi Comprehensive Cancer Center, Washington, USA
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34
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Liu C, Si X, Yan S, Zhao X, Qian X, Ying W, Zhao L. Development of the C12Im-Cl-assisted method for rapid sample preparation in proteomic application. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2021; 13:776-781. [PMID: 33492312 DOI: 10.1039/d0ay02079f] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods used prior to MS, including protein extraction and digestion, remain bottlenecks in the large-scale clinical application of proteomics. Ionic liquids, composed entirely of ions, have high solubility in various solvents. In this study, the effects of the cationic surfactant 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) on protein digestion were evaluated for clinical proteomic applications. C12Im-Cl was compatible with trypsin and reduced the protein digestion time from 16 h to 1 h. Residual C12Im-Cl was easily removed with a strong anion exchange membrane before MS. We evaluated the performance of C12Im-Cl extraction and rapid protein digestion using formalin-fixed paraffin-embedded liver cancer tissues. The number of proteins and peptides identified was nearly equal to that identified by the traditional filter-aided sample preparation method (2705 vs. 2739 and 16 682 vs. 17 214). In general, the C12Im-Cl-aided rapid sample preparation method is promising for proteomic applications.
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Affiliation(s)
- Chang Liu
- Beijing Key Laboratory of Environmental and Viral Oncology, Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China.
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35
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Mallach A, Gobom J, Zetterberg H, Hardy J, Piers TM, Wray S, Pocock JM. The influence of the R47H triggering receptor expressed on myeloid cells 2 variant on microglial exosome profiles. Brain Commun 2021; 3:fcab009. [PMID: 34704019 PMCID: PMC8244649 DOI: 10.1093/braincomms/fcab009] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 12/03/2020] [Accepted: 12/04/2020] [Indexed: 01/08/2023] Open
Abstract
Variants in the triggering receptor expressed on myeloid cells 2 gene are linked with an increased risk of dementia, in particular the R47Hhet triggering receptor expressed on myeloid cells 2 variant is linked to late-onset Alzheimer's disease. Using human induced pluripotent stem cells-derived microglia, we assessed whether variations in the dynamics of exosome secretion, including their components, from these cells might underlie some of this risk. We found exosome size was not altered between common variant controls and R47Hhet variants, but the amount and constitution of exosomes secreted were different. Exosome quantities were rescued by incubation with an ATP donor or with lipids via a phosphatidylserine triggering receptor expressed on myeloid cells 2 ligand. Following a lipopolysaccharide or phagocytic cell stimulus, exosomes from common variant and R47Hhet microglia were found to contain cytokines, chemokines, APOE and triggering receptor expressed on myeloid cells 2. Differences were observed in the expression of CCL22, IL-1β and triggering receptor expressed on myeloid cells 2 between common variant and R47Hhet derived exosomes. Furthermore unlike common variant-derived exosomes, R47Hhet exosomes contained additional proteins linked to negative regulation of transcription and metabolic processes. Subsequent addition of exosomes to stressed neurones showed R47Hhet-derived exosomes to be less protective. These data have ramifications for the responses of microglia in Alzheimer's disease and may point to further targets for therapeutic intervention.
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Affiliation(s)
- Anna Mallach
- Department of Neuroinflammation, Queen Square Institute of Neurology, University College London, London WC1N 1PJ, UK
| | - Johan Gobom
- Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, University of Gothenburg, S-431 80, Mölndal, Sweden
- Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, S-431 80, Mölndal, Sweden
| | - Henrik Zetterberg
- Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, University of Gothenburg, S-431 80, Mölndal, Sweden
- Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, S-431 80, Mölndal, Sweden
- Department of Neurodegenerative Disease, University College London Queen Square Institute of Neurology, London WC1N 1PJ, UK
- Department of Neurodegenerative Disease, University College London Queen Square Institute of Neurology, London WC1N 3BG, UK
- UK Dementia Research Institute University College London, London WC1E 6BT, UK
| | - John Hardy
- Department of Neurodegenerative Disease, University College London Queen Square Institute of Neurology, London WC1N 1PJ, UK
- Department of Neurodegenerative Disease, University College London Queen Square Institute of Neurology, London WC1N 3BG, UK
- UK Dementia Research Institute University College London, London WC1E 6BT, UK
- Reta Lila Weston Institute, UCL Queen Square Institute of Neurology, London WC1N 1PJ, UK
- NIHR University College London Hospitals Biomedical Research Centre, London, UK
- Institute for Advanced Study, The Hong Kong University of Science and Technology, Hong Kong SAR, China
| | - Thomas M Piers
- Department of Neuroinflammation, Queen Square Institute of Neurology, University College London, London WC1N 1PJ, UK
| | - Selina Wray
- Department of Neurodegenerative Disease, University College London Queen Square Institute of Neurology, London WC1N 1PJ, UK
- Department of Neurodegenerative Disease, University College London Queen Square Institute of Neurology, London WC1N 3BG, UK
| | - Jennifer M Pocock
- Department of Neuroinflammation, Queen Square Institute of Neurology, University College London, London WC1N 1PJ, UK
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Abstract
Protocols are described for preparation of various types of samples for differential protein expression proteomics using nano liquid chromatography-tandem mass spectrometry (nLCMS/MS). Fresh frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, cells, pull-downs, immunoprecipitations, and plasma or serum can be processed. For transplantation studies, it should be noted that samples must be comparable; thus, similar cells or organ systems from the same species can be compared when only a minority of the proteins change expression levels. For xenotransplantation studies, caution should be taken, as different species possess proteins with differences in primary structure. The sample preparation technique is based on spin filter devices using sodium deoxycholate (SDC) as detergent for extraction of proteins. The detergent is removed using phase extraction with ethyl acetate, and samples can be used for label-free quantification (LFQ) analysis or peptides can be labeled with tandem mass tags (TMT).
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Affiliation(s)
- Bent Honoré
- Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
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37
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Davalieva K, Kiprijanovska S, Dimovski A, Rosoklija G, Dwork AJ. Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues. J Proteomics 2021; 235:104117. [PMID: 33453434 DOI: 10.1016/j.jprot.2021.104117] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 01/05/2021] [Accepted: 01/05/2021] [Indexed: 10/22/2022]
Abstract
The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64-69% overlap, depletion of proteins >70 kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies. SIGNIFICANCE: Here we analyzed the performance of two sample preparation methods for shotgun proteomic analysis of FFPE tissues to give a comprehensive overview of the obtained proteomes and the resemblance to its matching fresh frozen counterparts. These findings give us better understanding towards competent proteomics analysis of FFPE tissues. It is hoped that it will encourage further assessments of available protocols before establishing the most effective protocol for shotgun proteomic FFPE tissue analysis.
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Affiliation(s)
- Katarina Davalieva
- Research Centre for Genetic Engineering and Biotechnology "Georgi D Efremov", Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, North Macedonia.
| | - Sanja Kiprijanovska
- Research Centre for Genetic Engineering and Biotechnology "Georgi D Efremov", Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, North Macedonia
| | - Aleksandar Dimovski
- Research Centre for Genetic Engineering and Biotechnology "Georgi D Efremov", Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, North Macedonia; Faculty of Pharmacy, University "St. Cyril and Methodius", 50ta Divizija 6, 1000 Skopje, North Macedonia
| | - Gorazd Rosoklija
- Department of Psychiatry, Columbia University, New York, USA; Molecular Imaging and Neuropathology Division, New York State Psychiatric Institute, 1051 Riverside Drive, Unit 42, New York, NY 10032, USA
| | - Andrew J Dwork
- Department of Psychiatry, Columbia University, New York, USA; Molecular Imaging and Neuropathology Division, New York State Psychiatric Institute, 1051 Riverside Drive, Unit 42, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, USA
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38
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Yaman ME, Kayili HM, Albayrak M, Kadioglu Y, Salih B. Differential N-glycosylation profiling of formalin-fixed paraffin-embedded (FFPE) invasive ductal carcinoma tissues using MALDI-TOF-MS. Mol Omics 2021; 17:394-404. [PMID: 33735360 DOI: 10.1039/d0mo00150c] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Invasive ductal carcinoma (IDC) is the most common type of breast cancer. As dynamic changes of the glycome are closely associated with complex diseases, they have become a focal point of cancer research involving predictive and prognostic markers. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are representative of the tumor environment and are thus utilized in studies on cancer related research and biomarker discovery. Further studies on differential N-glycosylation profiling of IDC cancer tissues are necessary in order to understand the biological role of glycans in cancer and to evaluate their predictive ability. In this study, matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)-based analyses were conducted for determining differential N-glycosylation patterns of IDC. Two different derivatization methods, namely, 2-aminobenzoic acid (2-AA) labeling and linkage-specific sialic acid esterification, were used for the analysis of N-glycans. Forty-seven 2-AA labeled and fifty ethyl esterified N-glycans were identified by MALDI-MS. In statistical analyses conducted for 2-AA-labeled N-glycans, the relative amounts of 32 N-glycans and prevalence of 15 N-glycan traits showed significant (p < 0.05) differences between cancer and normal tissues; and in such analyses for the ethyl-esterified N-glycans, the relative amounts of 27 N-glycans and prevalence of 17 N-glycan traits showed significant (p < 0.05) differences between them. It was found that mainly high mannose N-glycans, including H5N2, H6N2, and H7N2, and two fucosylated compositions (H3N3F1 and H5N5F1) showed strong discrimination between IDC and controls. In addition, compared with the controls, high mannose N-glycans were observed to be up-regulated in IDC whereas bisecting N-glycans were down-regulated.
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Affiliation(s)
- Mehmet Emrah Yaman
- Atatürk University, Faculty of Pharmacy, Department of Analytical Chemistry, Erzurum, 25240, Turkey
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39
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Kuras M, Woldmar N, Kim Y, Hefner M, Malm J, Moldvay J, Döme B, Fillinger J, Pizzatti L, Gil J, Marko-Varga G, Rezeli M. Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives. J Proteome Res 2020; 20:1027-1039. [PMID: 33301673 DOI: 10.1021/acs.jproteome.0c00850] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Well-characterized archival formalin-fixed paraffin-embedded (FFPE) tissues are of much value for prospective biomarker discovery studies, and protocols that offer high throughput and good reproducibility are essential in proteomics. Therefore, we implemented efficient paraffin removal and protein extraction from FFPE tissues followed by an optimized two-enzyme digestion using suspension trapping (S-Trap). The protocol was then combined with TMTpro 16plex labeling and applied to lung adenocarcinoma patient samples. In total, 9585 proteins were identified, and proteins related to the clinical outcome were detected. Because acetylation is known to play a major role in cancer development, a fast on-trap acetylation protocol was developed for studying endogenous lysine acetylation, which allows identification and localization of the lysine acetylation together with quantitative comparison between samples. We demonstrated that FFPE tissues are equivalent to frozen tissues to study the degree of acetylation between patients. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with isobaric labeling and for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives. The MS proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020157, PXD021986, and PXD021964.
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Affiliation(s)
- Magdalena Kuras
- Div. Clinical Chemistry, Dept. of Translational Medicine, Lund University, Skåne University Hospital Malmö, Malmö 20502, Sweden
| | - Nicole Woldmar
- Div. Clinical Protein Science & Imaging, Dept. of Clinical Sciences (Lund) and Dept. of Biomedical Engineering, Lund University, Lund 22100, Sweden.,Laboratory of Molecular Biology and Blood Proteomics - LADETEC, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-598, Brazil
| | - Yonghyo Kim
- Div. Clinical Protein Science & Imaging, Dept. of Clinical Sciences (Lund) and Dept. of Biomedical Engineering, Lund University, Lund 22100, Sweden
| | - Max Hefner
- Div. Clinical Protein Science & Imaging, Dept. of Clinical Sciences (Lund) and Dept. of Biomedical Engineering, Lund University, Lund 22100, Sweden
| | - Johan Malm
- Div. Clinical Chemistry, Dept. of Translational Medicine, Lund University, Skåne University Hospital Malmö, Malmö 20502, Sweden
| | - Judit Moldvay
- Dept. of Pulmonology, National Korányi Institute of Pulmonology, Semmelweis University, Budapest 1085, Hungary
| | - Balázs Döme
- Dept. of Pulmonology, National Korányi Institute of Pulmonology, Semmelweis University, Budapest 1085, Hungary.,Dept. of Thoracic Surgery, National Institute of Oncology, Semmelweis University, Budapest 1085, Hungary.,Div. of Thoracic Surgery, Dept. of Surgery, Comprehensive Cancer Center Vienna, Medical University Vienna, Vienna 1090, Austria
| | - János Fillinger
- Dept. of Pulmonology, National Korányi Institute of Pulmonology, Semmelweis University, Budapest 1085, Hungary.,Dept. of Thoracic Surgery, National Institute of Oncology, Semmelweis University, Budapest 1085, Hungary
| | - Luciana Pizzatti
- Laboratory of Molecular Biology and Blood Proteomics - LADETEC, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-598, Brazil
| | - Jeovanis Gil
- Div. Clinical Protein Science & Imaging, Dept. of Clinical Sciences (Lund) and Dept. of Biomedical Engineering, Lund University, Lund 22100, Sweden
| | - György Marko-Varga
- Div. Clinical Protein Science & Imaging, Dept. of Clinical Sciences (Lund) and Dept. of Biomedical Engineering, Lund University, Lund 22100, Sweden
| | - Melinda Rezeli
- Div. Clinical Protein Science & Imaging, Dept. of Clinical Sciences (Lund) and Dept. of Biomedical Engineering, Lund University, Lund 22100, Sweden
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40
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Vainer ED, Kania-Almog J, Zatara G, Levin Y, Vainer GW. Novel Proteome Extraction Method Illustrates a Conserved Immunological Signature of MSI-H Colorectal Tumors. Mol Cell Proteomics 2020; 19:1619-1631. [PMID: 32641473 PMCID: PMC8015011 DOI: 10.1074/mcp.ra120.002152] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2020] [Revised: 06/29/2020] [Indexed: 11/14/2022] Open
Abstract
Using a simple, environment friendly proteome extraction (TOP), we were able to optimize the analysis of clinical samples. Using our TOP method we analyzed a clinical cohort of microsatellite stable (MSS) and unstable (MSI-H) colorectal carcinoma (CRC). We identified a tumor cell specific, STAT1-centered, immune signature expressed by the MSI-H tumor cells. We then showed that long, but not short, exposure to Interferon-γ induces a similar signature in vitro We identified 10 different temporal protein expression patterns, classifying the Interferon-γ protein temporal regulation in CRC. Our data sheds light on the changes that tumor cells undergo under long-term immunological pressure in vivo, the importance of STAT proteins in specific biological scenarios. The data generated could help find novel clinical biomarkers and therapeutic approaches.
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Affiliation(s)
- Elez D Vainer
- Department of Gastroenterology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
| | - Juliane Kania-Almog
- Tel Aviv Sourasky Medical Center, Sackler School of Medicine, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
| | - Ghadeer Zatara
- Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
| | - Yishai Levin
- De Botton Institute for Protein Profiling, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, Israel
| | - Gilad W Vainer
- Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
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41
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Coscia F, Doll S, Bech JM, Schweizer L, Mund A, Lengyel E, Lindebjerg J, Madsen GI, Moreira JM, Mann M. A streamlined mass spectrometry-based proteomics workflow for large-scale FFPE tissue analysis. J Pathol 2020; 251:100-112. [PMID: 32154592 DOI: 10.1002/path.5420] [Citation(s) in RCA: 121] [Impact Index Per Article: 24.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2019] [Revised: 03/02/2020] [Accepted: 03/04/2020] [Indexed: 12/15/2022]
Abstract
Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Affiliation(s)
- Fabian Coscia
- Clinical Proteomics Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.,Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Sophia Doll
- Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Jacob Mathias Bech
- Section for Molecular Disease Biology, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Lisa Schweizer
- Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Andreas Mund
- Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Ernst Lengyel
- Department of Obstetrics and Gynecology, Section of Gynecologic Oncology, University of Chicago, Chicago, IL, USA
| | - Jan Lindebjerg
- Lillebaelt Hospital, Vejle Hospital, Department of Pathology, Vejle, Denmark
| | | | - José Ma Moreira
- Section for Molecular Disease Biology, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Matthias Mann
- Clinical Proteomics Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.,Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
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42
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Zeneyedpour L, Stingl C, Dekker LJM, Mustafa DAM, Kros JM, Luider TM. Phosphorylation Ratio Determination in Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Tissue with Targeted Mass Spectrometry. J Proteome Res 2020; 19:4179-4190. [PMID: 32811146 DOI: 10.1021/acs.jproteome.0c00354] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. These are, therefore, the most accessible research sources in pathology archives. In this study we investigated whether we can apply a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples in a sensitive and reproducible way. The feasibility of this technical approach was demonstrated for normal brain and glioblastoma multiforme tissues. Two methods were used: PRM measurement of a tryptic digest without phosphopeptide enrichment (Direct-PRM) and after Fe-NTA phosphopeptide enrichment (Fe-NTA-PRM). With these two methods, the phosphorylation ratio could be determined for four selected peptide pairs that originate from neuroblast differentiation-associated protein (AHNAK S5448-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), eukaryotic translation initiation factor 4B (EIF4B S93-p), and epidermal growth factor receptor (EGFR S1166-p). In normal brain FFPE tissues, the Fe-NTA-PRM method enabled the quantification of targeted phosphorylated peptides with high reproducibility (CV < 14%). Our results indicate that formalin fixation does not impede relative quantification of a phospho-site and its phosphorylation ratio in FFPE tissues. The developed workflow combining these methods opens ways to study archival FFPE tissues for phosphorylation ratio determination in proteins.
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Affiliation(s)
- Lona Zeneyedpour
- Department of Neurology, Erasmus MC, 3000 CA Rotterdam, The Netherlands
| | - Christoph Stingl
- Department of Neurology, Erasmus MC, 3000 CA Rotterdam, The Netherlands
| | | | - Dana A M Mustafa
- Department of Pathology, Erasmus MC, 3000 CA Rotterdam, The Netherlands
| | - Johan M Kros
- Department of Pathology, Erasmus MC, 3000 CA Rotterdam, The Netherlands
| | - Theo M Luider
- Department of Neurology, Erasmus MC, 3000 CA Rotterdam, The Netherlands
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43
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Buczak K, Kirkpatrick JM, Truckenmueller F, Santinha D, Ferreira L, Roessler S, Singer S, Beck M, Ori A. Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry. Nat Protoc 2020; 15:2956-2979. [PMID: 32737464 DOI: 10.1038/s41596-020-0356-y] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2019] [Accepted: 05/14/2020] [Indexed: 01/09/2023]
Abstract
Bottom-up mass spectrometry-based proteomics relies on protein digestion and peptide purification. The application of such methods to broadly available clinical samples such as formalin-fixed and paraffin-embedded (FFPE) tissues requires reversal of chemical crosslinking and the removal of reagents that are incompatible with mass spectrometry. Here, we describe in detail a protocol that combines tissue disruption by ultrasonication, heat-induced antigen retrieval and two alternative methods for efficient detergent removal to enable quantitative proteomic analysis of limited amounts of FFPE material. To show the applicability of our approach, we used hepatocellular carcinoma (HCC) as a model system. By combining the described protocol with laser-capture microdissection, we were able to quantify the intra-tumor heterogeneity of a tumor specimen on the proteome level using a single slide with tissue of 10-µm thickness. We also demonstrate broader applicability to other tissues, including human gallbladder and heart. The procedure described in this protocol can be completed within 8 d.
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Affiliation(s)
- Katarzyna Buczak
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.,Biozentrum, University of Basel, Basel, Switzerland
| | - Joanna M Kirkpatrick
- Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany.,Proteomics Science Technology Platform, The Francis Crick Institute, London, UK
| | | | - Deolinda Santinha
- Center for Neuroscience and Cell Biology and Faculty of Medicine, University of Coimbra, Coimbra, Portugal
| | - Lino Ferreira
- Center for Neuroscience and Cell Biology and Faculty of Medicine, University of Coimbra, Coimbra, Portugal
| | - Stephanie Roessler
- Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany
| | - Stephan Singer
- Institute of Pathology, University Hospital Tuebingen, Tuebingen, Germany
| | - Martin Beck
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. .,Department of Molecular Sociology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
| | - Alessandro Ori
- Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany.
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44
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Bayer M, Angenendt L, Schliemann C, Hartmann W, König S. Are formalin-fixed and paraffin-embedded tissues fit for proteomic analysis? JOURNAL OF MASS SPECTROMETRY : JMS 2020; 55:e4347. [PMID: 30828905 DOI: 10.1002/jms.4347] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Revised: 02/12/2019] [Accepted: 02/25/2019] [Indexed: 06/09/2023]
Abstract
Formalin-fixed and paraffin-embedded (FFPE)-tissue archives are potential treasure troves in the search for clinically interesting specimens. However, while the FFPE-treatment provides excellent conservation of the three-dimensional structure of the tissue and prevents degradation over decades, it also introduces numerous nonspecific and irreversible protein modifications. In this study, we have evaluated several published workflows for FFPE-tissue by fit-for-purpose proteomics technologies. We demonstrate that many protein modifications and cross-links remain after treatment and conclude that the proteomics of FFPE-tissue is of value, but clear-cut limitations must be kept in mind. The analysis of abundant proteins in FFPE is straightforward, but confident identification of low-level proteins and/or biologically relevant modifications is seriously hampered by the FFPE-treatment. Peptide assignment should only be performed on high-quality spectra, even if this is at the cost of lower numbers of protein IDs. As Yergey and Coorssen stated in 2015: "Data quality is considered the primary criterion, and we thus emphasize that the standards of Analytical Chemistry must apply throughout any proteomic analysis."
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Affiliation(s)
- Malte Bayer
- Core Unit Proteomics, Interdisciplinary Center for Clinical Research, Medical Faculty, University of Münster, Münster, Germany
| | - Linus Angenendt
- Department of Medicine A, Haematology and Oncology, University Hospital Münster, Münster, Germany
| | - Christoph Schliemann
- Department of Medicine A, Haematology and Oncology, University Hospital Münster, Münster, Germany
| | - Wolfgang Hartmann
- Gerhard-Domagk-Institute of Pathology, University Hospital Münster, Münster, Germany
| | - Simone König
- Core Unit Proteomics, Interdisciplinary Center for Clinical Research, Medical Faculty, University of Münster, Münster, Germany
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45
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Establishment and validation of highly accurate formalin-fixed paraffin-embedded quantitative proteomics by heat-compatible pressure cycling technology using phase-transfer surfactant and SWATH-MS. Sci Rep 2020; 10:11271. [PMID: 32647189 PMCID: PMC7347883 DOI: 10.1038/s41598-020-68245-2] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2020] [Accepted: 06/22/2020] [Indexed: 11/08/2022] Open
Abstract
The purpose of this study was to establish a quantitative proteomic method able to accurately quantify pathological changes in the protein expression levels of not only non-membrane proteins, but also membrane proteins, using formalin-fixed paraffin-embedded (FFPE) samples. Protein extraction from FFPE sections of mouse liver was increased 3.33-fold by pressure cycling technology (PCT) and reached the same level as protein extraction from frozen sections. After PCT-assisted processing of FFPE liver samples followed by SWATH-MS-based comprehensive quantification, the peak areas of 88.4% of peptides agreed with those from matched fresh samples within a 1.5-fold range. For membrane proteins, this percentage was remarkably increased from 49.1 to 93.8% by PCT. Compared to the conventional method using urea buffer, the present method using phase-transfer surfactant (PTS) buffer at 95 °C showed better agreement of peptide peak areas between FFPE and fresh samples. When our method using PCT and PTS buffer at 95 °C was applied to a bile duct ligation (BDL) disease model, the BDL/control expression ratios for 80.0% of peptides agreed within a 1.2-fold range between FFPE and fresh samples. This heat-compatible FFPE-PCT-SWATH proteomics technology using PTS is suitable for quantitative studies of pathological molecular mechanisms and biomarker discovery utilizing widely available FFPE samples.
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46
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Huang P, Kong Q, Gao W, Chu B, Li H, Mao Y, Cai Z, Xu R, Tian R. Spatial proteome profiling by immunohistochemistry-based laser capture microdissection and data-independent acquisition proteomics. Anal Chim Acta 2020; 1127:140-148. [PMID: 32800117 DOI: 10.1016/j.aca.2020.06.049] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2020] [Revised: 06/18/2020] [Accepted: 06/20/2020] [Indexed: 12/11/2022]
Abstract
Understanding the tumor heterogeneity through spatially resolved proteome profiling is important for biomedical research and clinical application. Laser capture microdissection (LCM) is a powerful technology for exploring local cell populations without losing spatial information. Conventionally, tissue sections are stained with hematoxylin and eosin (H&E) for cell-type identification before LCM. However, it generally requires experienced pathologists to distinguish different cell types, which limits the application of LCM to broad cancer research field. Here, we designed an immunohistochemistry (IHC)-based workflow for cell type-resolved proteome analysis of tissue samples. Firstly, targeted cell type was marked by IHC using antibody targeting cell-type specific marker to improve accuracy and efficiency of LCM. Secondly, to increase protein recovery from chemically crosslinked IHC tissues, we optimized a decrosslinking procedure to seamlessly combine with the integrated spintip-based sample preparation technology SISPROT. This newly developed approach, termed IHC-SISPROT, has comparable performance as H&E staining-based proteomic analysis. High sensitivity and reproducibility of IHC-SISPROT were achieved by combining with data independent acquisition proteomics. More than 3500 proteins were identified from only 0.2 mm2 and 12 μm thickness of hepatocellular carcinoma (HCC) tissue section. Furthermore, using 5 mm2 and 12 μm thickness of HCC tissue section, 6660 and 6052 protein groups were quantified from cancer cells and cancer-associated fibroblasts (CAFs) by the IHC-SISPROT workflow. Bioinformatic analysis revealed the enrichment of cell type-specific ligands and receptors and potentially new communications between cancer cells and CAFs by these signaling proteins. Therefore, IHC-SISPROT is a sensitive and accurate proteomic approach for spatial profiling of cell type-specific proteome from tissues.
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Affiliation(s)
- Peiwu Huang
- Department of Chemistry, Southern University of Science and Technology, Shenzhen, 518055, China; State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, SAR, China
| | - Qian Kong
- Department of Chemistry, Southern University of Science and Technology, Shenzhen, 518055, China; State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, SAR, China
| | - Weina Gao
- Department of Chemistry, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Bizhu Chu
- Department of Chemistry, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Hua Li
- State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, SAR, China; SUSTech Core Research Facilities, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Yiheng Mao
- Department of Chemistry, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Zongwei Cai
- State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, SAR, China
| | - Ruilian Xu
- Shenzhen People's Hospital, The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, 518020, China
| | - Ruijun Tian
- Department of Chemistry, Southern University of Science and Technology, Shenzhen, 518055, China; Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen, 518055, China.
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47
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Platform independent protein-based cell-of-origin subtyping of diffuse large B-cell lymphoma in formalin-fixed paraffin-embedded tissue. Sci Rep 2020; 10:7876. [PMID: 32398793 PMCID: PMC7217957 DOI: 10.1038/s41598-020-64212-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Accepted: 04/09/2020] [Indexed: 01/03/2023] Open
Abstract
Diffuse large B-cell lymphoma (DLBCL) is commonly classified by gene expression profiling according to its cell of origin (COO) into activated B-cell (ABC)-like and germinal center B-cell (GCB)-like subgroups. Here we report the application of label-free nano-liquid chromatography - Sequential Window Acquisition of all THeoretical fragment-ion spectra - mass spectrometry (nanoLC-SWATH-MS) to the COO classification of DLBCL in formalin-fixed paraffin-embedded (FFPE) tissue. To generate a protein signature capable of predicting Affymetrix-based GCB scores, the summed log2-transformed fragment ion intensities of 780 proteins quantified in a training set of 42 DLBCL cases were used as independent variables in a penalized zero-sum elastic net regression model with variable selection. The eight-protein signature obtained showed an excellent correlation (r = 0.873) between predicted and true GCB scores and yielded only 9 (21.4%) minor discrepancies between the three classifications: ABC, GCB, and unclassified. The robustness of the model was validated successfully in two independent cohorts of 42 and 31 DLBCL cases, the latter cohort comprising only patients aged >75 years, with Pearson correlation coefficients of 0.846 and 0.815, respectively, between predicted and NanoString nCounter based GCB scores. We further show that the 8-protein signature is directly transferable to both a triple quadrupole and a Q Exactive quadrupole-Orbitrap mass spectrometer, thus obviating the need for proprietary instrumentation and reagents. This method may therefore be used for robust and competitive classification of DLBCLs on the protein level.
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48
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Wang X, Cheng C, Li Q, Zhang K, Lou Q, Li J, Chen J. Multi-omics analysis revealed that MAPK signaling and flavonoid metabolic pathway contributed to resistance against Meloidogyne incognita in the introgression line cucumber. J Proteomics 2020; 220:103675. [PMID: 32004728 DOI: 10.1016/j.jprot.2020.103675] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2019] [Revised: 01/07/2020] [Accepted: 01/28/2020] [Indexed: 01/04/2023]
Abstract
Inhibiting giant cells (GCs) development is an important characteristic of introgression line cucumber IL10-1 against Meloidogyne incognita proved by our previous study, but the systematic regulatory pathways were unknown. To reveal the regulation pathways more comprehensively, in the current study, we performed a joint analysis of RNA-Seq and lable-free quantitative proteomics between the IL10-1 (resistant) and the CC3 (susceptible cucumber) after inoculation with M. incognita. The results indicated that flavonoid biosynthesis pathway was specifically enriched in IL10-1. And protein species of Csa5P590220 and Csa5P589940 associated with flavonoid biosynthesis were highly translated in IL10-1 compared with these in CC3 at 3 days post inoculation (dpi), which would resulted in the excess of flavonoids in IL10-1 roots. In addition, phosphoproteomic analysis found that phosphorylated protein species involved in MAPK signaling cascade were enhanced in IL10-1, while they were inhibited in CC3 at 3 dpi. Accordingly, we speculate that the enhanced MAPK cascade signaling plays an important role in signal transduction for IL10-1 regulating the flavonoid biosynthesis. Knowledge from the study provide important regulatory pathways and protein species of introgression line cucumber against M. incognita, which will help in efforts to improve the recognition of the resistance mechanism of plants against nematode. SIGNIFICANCE: The current approach of joint analysis in transcription level, protein level and protein phosphorylation level more comprehensively revealed the different expression patterns at the molecular level of resistant and susceptible cucumber after inoculation with M. incognita. Based on the different expression patterns, we explore the pathway of resistance regulation of resistant cucumber IL10-1. Moreover, our results are helpful for the discovery of key genes and then apply them to M. incognita-resistance breeding.
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Affiliation(s)
- Xing Wang
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.
| | - Chunyan Cheng
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.
| | - Qingrong Li
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.
| | - Kaijing Zhang
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.
| | - Qunfeng Lou
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.
| | - Ji Li
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.
| | - Jinfeng Chen
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.
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49
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Yin B, Mendez R, Zhao XY, Rakhit R, Hsu KL, Ewald SE. Automated Spatially Targeted Optical Microproteomics (autoSTOMP) to Determine Protein Complexity of Subcellular Structures. Anal Chem 2020; 92:2005-2010. [PMID: 31869197 DOI: 10.1021/acs.analchem.9b04396] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Spatially targeted optical microproteomics (STOMP) is a method to study region-specific protein complexity in primary cells and tissue samples. STOMP uses a confocal microscope to visualize structures of interest and to tag the proteins within those structures by a photodriven cross-linking reaction so that they can be affinity purified and identified by mass spectrometry (eLife 2015, 4, e09579). However, the use of a custom photo-cross-linker and the requirement for extensive user intervention during sample tagging have posed barriers to the utilization of STOMP. To address these limitations, we built automated STOMP (autoSTOMP) which uses a customizable code in SikuliX to coordinate image capture and cross-linking functions in Zeiss Zen Black with image processing in FIJI. To increase protocol accessibility, we implemented a commercially available biotin-benzophenone photo-cross-linking and purification protocol. Here we demonstrate that autoSTOMP can efficiently label, purify, and identify proteins belonging to 1-2 μm structures in primary human foreskin fibroblasts or mouse bone marrow-derived dendritic cells infected with the protozoan parasite Toxoplasma gondii (Tg). AutoSTOMP can easily be adapted to address a range of research questions using Zeiss Zen Black microscopy systems and LC-MS protocols that are standard in many research cores.
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Affiliation(s)
- Bocheng Yin
- Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center , University of Virginia School of Medicine , Charlottesville , Virginia 22908-0395 , United States
| | - Roberto Mendez
- Department of Chemistry , University of Virginia , Charlottesville , Virginia 22904-4132 , United States
| | - Xiao-Yu Zhao
- Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center , University of Virginia School of Medicine , Charlottesville , Virginia 22908-0395 , United States
| | - Rishi Rakhit
- Mitokinin Inc , 953 Indiana Street , San Francisco , California 94107-3007 , United States
| | - Ku-Lung Hsu
- Department of Chemistry , University of Virginia , Charlottesville , Virginia 22904-4132 , United States
| | - Sarah E Ewald
- Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center , University of Virginia School of Medicine , Charlottesville , Virginia 22908-0395 , United States
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50
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Affiliation(s)
| | | | - Ronghu Wu
- School of Chemistry and Biochemistry and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
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