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Koh JMS, Sykes EK, Rukhaya J, Anees A, Zhong Q, Jackson C, Panizza BJ, Reddel RR, Balleine RL, Hains PG, Robinson PJ. The effect of storage time and temperature on the proteomic analysis of FFPE tissue sections. Clin Proteomics 2025; 22:5. [PMID: 39910438 DOI: 10.1186/s12014-025-09529-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2024] [Accepted: 01/22/2025] [Indexed: 02/07/2025] Open
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissues present an important resource for cancer proteomics. They are more readily available than fresh frozen (FF) tissues and can be stored at ambient temperature for decades. FFPE blocks are largely stable for long-term preservation of tumour histology, but the antigenicity of some proteins in FFPE sections degrades over time resulting in deteriorating performance of immunohistochemistry (IHC). It is not known whether FFPE sections that have previously been cut from blocks and used for liquid chromatography-mass spectrometry (LC-MS) analysis at a later time are affected by storage time or temperature. We determined the stability of FFPE sections stored at room temperature (RT) versus - 80 °C over 48 weeks. The stored sections were processed at different timepoints (n = 11) and compared to sections that were freshly cut from FFPE blocks at each timepoint (controls). A total of 297 sections (rat brain, kidney and liver stored at RT, - 80 °C or freshly cut) were tryptically digested and analysed on TripleTOF 6600 mass spectrometers in data-dependent acquisition (DDA) mode. Kidney and liver digests were also analysed in data-independent acquisition (DIA) mode. The number of proteins and peptides identified by DDA with ProteinPilot and some common post-translational modifications (PTMs) were unaffected by the storage time or temperature. Nine of the most common FFPE-associated modifications were quantified using DIA data and all were unaffected by storage time or temperature. Therefore, FFPE tissue sections are suitable for proteomic studies for at least 48 weeks from the time of sectioning.
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Affiliation(s)
- Jennifer M S Koh
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
| | - Erin K Sykes
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
| | - Jyoti Rukhaya
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
| | - Asim Anees
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
| | - Qing Zhong
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
| | - Christopher Jackson
- Department of Otolaryngology, Head and Neck Surgery, Princess Alexandra Hospital, Brisbane, QLD, Australia
- Queensland Head and Neck Cancer Centre, Princess Alexandra Hospital, Brisbane, QLD, Australia
- Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia
| | - Benedict J Panizza
- Department of Otolaryngology, Head and Neck Surgery, Princess Alexandra Hospital, Brisbane, QLD, Australia
- Queensland Head and Neck Cancer Centre, Princess Alexandra Hospital, Brisbane, QLD, Australia
- Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia
| | - Roger R Reddel
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
| | - Rosemary L Balleine
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
- Faculty of Medicine and Health, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia
| | - Peter G Hains
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
| | - Phillip J Robinson
- Faculty of Medicine and Health, ProCan®, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia.
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2
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Egbejiogu BC, Donnarumma F, Murray KK. Infrared Laser Ablation and Capture of Formalin-Fixed Paraffin-Embedded Tissue. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2024. [PMID: 39494617 DOI: 10.1021/jasms.4c00299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2024]
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissue is a ubiquitous and invaluable resource for biomedical research and clinical applications. However, FFPE tissue proteomics is challenging due to protein cross-linking and chemical modification. Laser ablation sampling allows precise removal of material from tissue sections with high spatial control and reproducibility for offline proteomics by liquid chromatography coupled with tandem mass spectrometry. In this work, we used a pulsed mid-infrared laser for microsampling of rat liver tissue for subsequent identification and quantification of proteins. It was found that more proteins were identified by FFPE tissue laser ablation sampling compared to fresh frozen (FF) tissue laser ablation sampling and that more proteins were identified by laser ablation than by manual dissection of FFPE tissue. In contrast to previous studies, no loss of hydrophilic proteins due to residual cross-linking was observed. The efficient capture of proteins by laser ablation microsampling is attributed to efficient laser breakup of the tissue which facilitates downstream processing of the proteins.
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Affiliation(s)
- Blessing C Egbejiogu
- Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States
| | - Fabrizio Donnarumma
- Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States
| | - Kermit K Murray
- Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States
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3
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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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4
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Faktor J, Kote S, Bienkowski M, Hupp TR, Marek-Trzonkowska N. Novel FFPE proteomics method suggests prolactin induced protein as hormone induced cytoskeleton remodeling spatial biomarker. Commun Biol 2024; 7:708. [PMID: 38851810 PMCID: PMC11162451 DOI: 10.1038/s42003-024-06354-8] [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: 01/20/2023] [Accepted: 05/20/2024] [Indexed: 06/10/2024] Open
Abstract
Robotically assisted proteomics provides insights into the regulation of multiple proteins achieving excellent spatial resolution. However, developing an effective method for spatially resolved quantitative proteomics of formalin fixed paraffin embedded tissue (FFPE) in an accessible and economical manner remains challenging. We introduce non-robotic In-insert FFPE proteomics approach, combining glass insert FFPE tissue processing with spatial quantitative data-independent mass spectrometry (DIA). In-insert approach identifies 450 proteins from a 5 µm thick breast FFPE tissue voxel with 50 µm lateral dimensions covering several tens of cells. Furthermore, In-insert approach associated a keratin series and moesin (MOES) with prolactin-induced protein (PIP) indicating their prolactin and/or estrogen regulation. Our data suggest that PIP is a spatial biomarker for hormonally triggered cytoskeletal remodeling, potentially useful for screening hormonally affected hotspots in breast tissue. In-insert proteomics represents an alternative FFPE processing method, requiring minimal laboratory equipment and skills to generate spatial proteotype repositories from FFPE tissue.
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Affiliation(s)
- Jakub Faktor
- International Centre for Cancer Vaccine Science, University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
| | - Sachin Kote
- International Centre for Cancer Vaccine Science, University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
| | - Michal Bienkowski
- Medical University of Gdansk, University of Gdansk, Mariana Smoluchowskiego 17, 80-214, Gdansk, Poland
| | - Ted R Hupp
- International Centre for Cancer Vaccine Science, University of Gdansk, Kladki 24, 80-822, Gdansk, Poland
| | - Natalia Marek-Trzonkowska
- International Centre for Cancer Vaccine Science, University of Gdansk, Kladki 24, 80-822, Gdansk, Poland
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5
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Schweizer L, Schaller T, Zwiebel M, Karayel Ö, Müller‐Reif JB, Zeng W, Dintner S, Nordmann TM, Hirschbühl K, Märkl B, Claus R, Mann M. Quantitative multiorgan proteomics of fatal COVID-19 uncovers tissue-specific effects beyond inflammation. EMBO Mol Med 2023; 15:e17459. [PMID: 37519267 PMCID: PMC10493576 DOI: 10.15252/emmm.202317459] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 07/11/2023] [Accepted: 07/14/2023] [Indexed: 08/01/2023] Open
Abstract
SARS-CoV-2 may directly and indirectly damage lung tissue and other host organs, but there are few system-wide, untargeted studies of these effects on the human body. Here, we developed a parallelized mass spectrometry (MS) proteomics workflow enabling the rapid, quantitative analysis of hundreds of virus-infected FFPE tissues. The first layer of response to SARS-CoV-2 in all tissues was dominated by circulating inflammatory molecules. Beyond systemic inflammation, we differentiated between systemic and true tissue-specific effects to reflect distinct COVID-19-associated damage patterns. Proteomic changes in the lungs resembled those of diffuse alveolar damage (DAD) in non-COVID-19 patients. Extensive organ-specific changes were also evident in the kidneys, liver, and lymphatic and vascular systems. Secondary inflammatory effects in the brain were related to rearrangements in neurotransmitter receptors and myelin degradation. These MS-proteomics-derived results contribute substantially to our understanding of COVID-19 pathomechanisms and suggest strategies for organ-specific therapeutic interventions.
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Affiliation(s)
- Lisa Schweizer
- Department of Proteomics and Signal TransductionMax Planck Institute of BiochemistryMartinsriedGermany
| | - Tina Schaller
- Pathology, Medical FacultyUniversity of AugsburgAugsburgGermany
| | - Maximilian Zwiebel
- Department of Proteomics and Signal TransductionMax Planck Institute of BiochemistryMartinsriedGermany
| | - Özge Karayel
- Department of Proteomics and Signal TransductionMax Planck Institute of BiochemistryMartinsriedGermany
- Present address:
Department of Physiological ChemistryGenentechSouth San FranciscoUSA
| | | | - Wen‐Feng Zeng
- Department of Proteomics and Signal TransductionMax Planck Institute of BiochemistryMartinsriedGermany
| | | | - Thierry M Nordmann
- Department of Proteomics and Signal TransductionMax Planck Institute of BiochemistryMartinsriedGermany
| | - Klaus Hirschbühl
- Hematology and Oncology, Medical FacultyUniversity of AugsburgAugsburgGermany
| | - Bruno Märkl
- Pathology, Medical FacultyUniversity of AugsburgAugsburgGermany
| | - Rainer Claus
- Pathology, Medical FacultyUniversity of AugsburgAugsburgGermany
- Hematology and Oncology, Medical FacultyUniversity of AugsburgAugsburgGermany
| | - Matthias Mann
- Department of Proteomics and Signal TransductionMax Planck Institute of BiochemistryMartinsriedGermany
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6
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Chen Z, Chuang D, Chen S, He Q, Tomlison BN, Cui J, Gu Z. Gelatin Zymography Can Be Performed on Fixed Brain Tissue. J Histochem Cytochem 2023; 71:481-493. [PMID: 37599425 PMCID: PMC10501363 DOI: 10.1369/00221554231194118] [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: 12/15/2022] [Accepted: 07/18/2023] [Indexed: 08/22/2023] Open
Abstract
Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481-493, 2023).
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Affiliation(s)
- Zhenzhou Chen
- Department of Neurosurgery, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Dennis Chuang
- Department of Pathology and Anatomical Sciences, School of Medicine, University of Missouri, Columbia, Missouri
- MultiCare Neuroscience & Sleep Medicine, Auburn, Washington
| | - Shanyan Chen
- Department of Pathology and Anatomical Sciences, School of Medicine, University of Missouri, Columbia, Missouri
| | - Qiwei He
- Department of Neurosurgery, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Brittany N. Tomlison
- Department of Pathology and Anatomical Sciences, School of Medicine, University of Missouri, Columbia, Missouri
| | - Jiankun Cui
- Department of Pathology and Anatomical Sciences, School of Medicine, University of Missouri, Columbia, Missouri
| | - Zezong Gu
- Department of Pathology and Anatomical Sciences, School of Medicine, University of Missouri, Columbia, Missouri
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7
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Davidson JM, Rayner SL, Liu S, Cheng F, Di Ieva A, Chung RS, Lee A. Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways. Int J Mol Sci 2023; 24:ijms24054283. [PMID: 36901711 PMCID: PMC10001664 DOI: 10.3390/ijms24054283] [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: 01/30/2023] [Revised: 02/16/2023] [Accepted: 02/19/2023] [Indexed: 02/24/2023] Open
Abstract
Proteomics offers vast potential for studying the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue; however, it presents challenges for proteomic analysis. In this study, we compared the efficiency of two different protein-extraction buffers on three post-mortem, formalin-fixed human brains. Equal amounts of extracted proteins were subjected to in-gel tryptic digestion and LC-MS/MS. Protein, peptide sequence, and peptide group identifications; protein abundance; and gene ontology pathways were analyzed. Protein extraction was superior using lysis buffer containing tris(hydroxymethyl)aminomethane hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 (TrisHCl, SDS, SDC, Triton X-100), which was then used for inter-regional analysis. Pre-frontal, motor, temporal, and occipital cortex tissues were analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways in different brain regions, suggesting commonalities in the molecular regulation of neuroanatomically-linked brain functions. Overall, we developed an optimized, robust, and efficient method for protein extraction from formalin-fixed human brain tissue for in-depth LFQ proteomics. We also demonstrate herein that this method is suitable for rapid and routine analysis to uncover molecular signaling pathways in the human brain.
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Affiliation(s)
- Jennilee M. Davidson
- Centre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, Australia
- Correspondence: (J.M.D.); (A.D.I.)
| | - Stephanie L. Rayner
- Centre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, Australia
| | - Sidong Liu
- Centre for Health Informatics, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW 2109, Australia
- Computational NeuroSurgery (CNS) Lab, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, Australia
| | - Flora Cheng
- Centre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, Australia
| | - Antonio Di Ieva
- Computational NeuroSurgery (CNS) Lab, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, Australia
- Correspondence: (J.M.D.); (A.D.I.)
| | - Roger S. Chung
- Centre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, Australia
| | - Albert Lee
- Centre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, Australia
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8
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Obi EN, Tellock DA, Thomas GJ, Veenstra TD. Biomarker Analysis of Formalin-Fixed Paraffin-Embedded Clinical Tissues Using Proteomics. Biomolecules 2023; 13:biom13010096. [PMID: 36671481 PMCID: PMC9855471 DOI: 10.3390/biom13010096] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2022] [Revised: 12/20/2022] [Accepted: 12/21/2022] [Indexed: 01/06/2023] Open
Abstract
The relatively recent developments in mass spectrometry (MS) have provided novel opportunities for this technology to impact modern medicine. One of those opportunities is in biomarker discovery and diagnostics. Key developments in sample preparation have enabled a greater range of clinical samples to be characterized at a deeper level using MS. While most of these developments have focused on blood, tissues have also been an important resource. Fresh tissues, however, are difficult to obtain for research purposes and require significant resources for long-term storage. There are millions of archived formalin-fixed paraffin-embedded (FFPE) tissues within pathology departments worldwide representing every possible tissue type including tumors that are rare or very small. Owing to the chemical technique used to preserve FFPE tissues, they were considered intractable to many newer proteomics techniques and primarily only useful for immunohistochemistry. In the past couple of decades, however, researchers have been able to develop methods to extract proteins from FFPE tissues in a form making them analyzable using state-of-the-art technologies such as MS and protein arrays. This review will discuss the history of these developments and provide examples of how they are currently being used to identify biomarkers and diagnose diseases such as cancer.
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9
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Baniasad M, Kim Y, Shaffer M, Sabag-Daigle A, Leleiwi I, Daly RA, Ahmer BMM, Wrighton KC, Wysocki VH. Optimization of proteomics sample preparation for identification of host and bacterial proteins in mouse feces. Anal Bioanal Chem 2022; 414:2317-2331. [PMID: 35106611 PMCID: PMC9393048 DOI: 10.1007/s00216-022-03885-z] [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: 09/22/2021] [Revised: 12/03/2021] [Accepted: 01/07/2022] [Indexed: 11/01/2022]
Abstract
Bottom-up proteomics is a powerful method for the functional characterization of mouse gut microbiota. To date, most of the bottom-up proteomics studies of the mouse gut rely on limited amounts of fecal samples. With mass-limited samples, the performance of such analyses is highly dependent on the protein extraction protocols and contaminant removal strategies. Here, protein extraction protocols (using different lysis buffers) and contaminant removal strategies (using different types of filters and beads) were systematically evaluated to maximize quantitative reproducibility and the number of identified proteins. Overall, our results recommend a protein extraction method using a combination of sodium dodecyl sulfate (SDS) and urea in Tris-HCl to yield the greatest number of protein identifications. These conditions led to an increase in the number of proteins identified from gram-positive bacteria, such as Firmicutes and Actinobacteria, which is a challenging task. Our analysis further confirmed these conditions led to the extraction of non-abundant bacterial phyla such as Proteobacteria. In addition, we found that, when coupled to our optimized extraction method, suspension trap (S-Trap) outperforms other contaminant removal methods by providing the most reproducible method while producing the greatest number of protein identifications. Overall, our optimized sample preparation workflow is straightforward and fast, and requires minimal sample handling. Furthermore, our approach does not require high amounts of fecal samples, a vital consideration in proteomics studies where mice produce smaller amounts of feces due to a particular physiological condition. Our final method provides efficient digestion of mouse fecal material, is reproducible, and leads to high proteomic coverage for both host and microbiome proteins.
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Affiliation(s)
- Maryam Baniasad
- Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA
| | - Yongseok Kim
- Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA
| | - Michael Shaffer
- Department of Soil and Crop Sciences, The Colorado State University, Fort Collins, CO, USA
| | - Anice Sabag-Daigle
- Department of Microbiology, The Ohio State University, Columbus, OH, USA
| | - Ikaia Leleiwi
- Department of Soil and Crop Sciences, The Colorado State University, Fort Collins, CO, USA
| | - Rebecca A Daly
- Department of Soil and Crop Sciences, The Colorado State University, Fort Collins, CO, USA
| | - Brian M M Ahmer
- Department of Microbiology, The Ohio State University, Columbus, OH, USA
| | - Kelly C Wrighton
- Department of Soil and Crop Sciences, The Colorado State University, Fort Collins, CO, USA
| | - Vicki H Wysocki
- Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA.
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10
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Mao Y, Wang X, Huang P, Tian R. Spatial proteomics for understanding the tissue microenvironment. Analyst 2021; 146:3777-3798. [PMID: 34042124 DOI: 10.1039/d1an00472g] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
The human body comprises rich populations of cells, which are arranged into tissues and organs with diverse functionalities. These cells exhibit a broad spectrum of phenotypes and are often organized as a heterogeneous but sophisticatedly regulated ecosystem - tissue microenvironment, inside which every cell interacts with and is reciprocally influenced by its surroundings through its life span. Therefore, it is critical to comprehensively explore the cellular machinery and biological processes in the tissue microenvironment, which is best exemplified by the tumor microenvironment (TME). The past decade has seen increasing advances in the field of spatial proteomics, the main purpose of which is to characterize the abundance and spatial distribution of proteins and their post-translational modifications in the microenvironment of diseased tissues. Herein, we outline the achievements and remaining challenges of mass spectrometry-based tissue spatial proteomics. Exciting technology developments along with important biomedical applications of spatial proteomics are highlighted. In detail, we focus on high-quality resources built by scalpel macrodissection-based region-resolved proteomics, method development of sensitive sample preparation for laser microdissection-based spatial proteomics, and antibody recognition-based multiplexed tissue imaging. In the end, critical issues and potential future directions for spatial proteomics are also discussed.
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Affiliation(s)
- Yiheng Mao
- School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, 150001, China. and Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China
| | - Xi Wang
- Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China and Shenzhen People's Hospital, The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518020, China
| | - Peiwu Huang
- Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China
| | - Ruijun Tian
- Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China
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11
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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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12
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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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13
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Flores Bueso Y, Walker SP, Tangney M. Characterization of FFPE-induced bacterial DNA damage and development of a repair method. Biol Methods Protoc 2020; 5:bpaa015. [PMID: 33072872 PMCID: PMC7548031 DOI: 10.1093/biomethods/bpaa015] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Revised: 07/15/2020] [Accepted: 07/18/2020] [Indexed: 01/13/2023] Open
Abstract
Formalin-fixed, paraffin-embedded (FFPE) specimens have huge potential as source material in the field of human microbiome research. However, the effects of FFPE processing on bacterial DNA remain uncharacterized. Any effects are relevant for microbiome studies, where DNA template is often minimal and sequences studied are not limited to one genome. As such, we aimed to both characterize this FFPE-induced bacterial DNA damage and develop strategies to reduce and repair this damage. Our analyses indicate that bacterial FFPE DNA is highly fragmented, a poor template for PCR, crosslinked and bears sequence artefacts derived predominantly from oxidative DNA damage. Two strategies to reduce this damage were devised – an optimized decrosslinking procedure reducing sequence artefacts generated by high-temperature incubation, and secondly, an in vitro reconstitution of the base excision repair pathway. As evidenced by whole genome sequencing, treatment with these strategies significantly increased fragment length, reduced the appearance of sequence artefacts and improved the sequencing readability of bacterial and mammalian FFPE DNA. This study provides a new understanding of the condition of bacterial DNA in FFPE specimens and how this impacts downstream analyses, in addition to a strategy to improve the sequencing quality of bacterial and possibly mammalian FFPE DNA.
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Affiliation(s)
- Yensi Flores Bueso
- CancerResearch@UCC, University College Cork, Cork, T12 XF62, Ireland.,SynBioCentre, University College Cork, Cork, T12 XF62, Ireland.,APC Microbiome Ireland, University College Cork, Cork, T12 YT20, Ireland
| | - Sidney P Walker
- CancerResearch@UCC, University College Cork, Cork, T12 XF62, Ireland.,SynBioCentre, University College Cork, Cork, T12 XF62, Ireland.,APC Microbiome Ireland, University College Cork, Cork, T12 YT20, Ireland
| | - Mark Tangney
- CancerResearch@UCC, University College Cork, Cork, T12 XF62, Ireland.,SynBioCentre, University College Cork, Cork, T12 XF62, Ireland.,APC Microbiome Ireland, University College Cork, Cork, T12 YT20, Ireland
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14
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Azimzadeh O, Azizova T, Merl-Pham J, Blutke A, Moseeva M, Zubkova O, Anastasov N, Feuchtinger A, Hauck SM, Atkinson MJ, Tapio S. Chronic Occupational Exposure to Ionizing Radiation Induces Alterations in the Structure and Metabolism of the Heart: A Proteomic Analysis of Human Formalin-Fixed Paraffin-Embedded (FFPE) Cardiac Tissue. Int J Mol Sci 2020; 21:ijms21186832. [PMID: 32957660 PMCID: PMC7555548 DOI: 10.3390/ijms21186832] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2020] [Revised: 09/14/2020] [Accepted: 09/14/2020] [Indexed: 12/23/2022] Open
Abstract
Epidemiological studies on workers employed at the Mayak plutonium enrichment plant have demonstrated an association between external gamma ray exposure and an elevated risk of ischemic heart disease (IHD). In a previous study using fresh-frozen post mortem samples of the cardiac left ventricle of Mayak workers and non-irradiated controls, we observed radiation-induced alterations in the heart proteome, mainly downregulation of mitochondrial and structural proteins. As the control group available at that time was younger than the irradiated group, we could not exclude age as a confounding factor. To address this issue, we have now expanded our study to investigate additional samples using archival formalin-fixed paraffin-embedded (FFPE) tissue. Importantly, the control group studied here is older than the occupationally exposed (>500 mGy) group. Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor.
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Affiliation(s)
- Omid Azimzadeh
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany; (N.A.); (M.J.A.); (S.T.)
- Correspondence: ; Tel.: +49-89-3187-3887
| | - Tamara Azizova
- Southern Urals Biophysics Institute (SUBI), Russian Federation, 456780 Ozyorsk, Russia; (T.A.); (M.M.); (O.Z.)
| | - Juliane Merl-Pham
- Helmholtz Zentrum München—German Research Centre for Environmental Health, Research Unit Protein Science, 80939 Munich, Germany; (J.M.-P.); (S.M.H.)
| | - Andreas Blutke
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Research Unit Analytical Pathology, 85764 Neuherberg, Germany; (A.B.); (A.F.)
| | - Maria Moseeva
- Southern Urals Biophysics Institute (SUBI), Russian Federation, 456780 Ozyorsk, Russia; (T.A.); (M.M.); (O.Z.)
| | - Olga Zubkova
- Southern Urals Biophysics Institute (SUBI), Russian Federation, 456780 Ozyorsk, Russia; (T.A.); (M.M.); (O.Z.)
| | - Natasa Anastasov
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany; (N.A.); (M.J.A.); (S.T.)
| | - Annette Feuchtinger
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Research Unit Analytical Pathology, 85764 Neuherberg, Germany; (A.B.); (A.F.)
| | - Stefanie M. Hauck
- Helmholtz Zentrum München—German Research Centre for Environmental Health, Research Unit Protein Science, 80939 Munich, Germany; (J.M.-P.); (S.M.H.)
| | - Michael J. Atkinson
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany; (N.A.); (M.J.A.); (S.T.)
- Chair of Radiation Biology, Technical University of Munich, 81675 Munich, Germany
| | - Soile Tapio
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany; (N.A.); (M.J.A.); (S.T.)
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15
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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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16
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Griesser E, Wyatt H, Ten Have S, Stierstorfer B, Lenter M, Lamond AI. Quantitative Profiling of the Human Substantia Nigra Proteome from Laser-capture Microdissected FFPE Tissue. Mol Cell Proteomics 2020; 19:839-851. [PMID: 32132230 PMCID: PMC7196589 DOI: 10.1074/mcp.ra119.001889] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2019] [Revised: 02/19/2020] [Indexed: 11/10/2022] Open
Abstract
Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (∼3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.
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Affiliation(s)
- Eva Griesser
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom; Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
| | - Hannah Wyatt
- Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
| | - Sara Ten Have
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom
| | - Birgit Stierstorfer
- Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
| | - Martin Lenter
- Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
| | - Angus I Lamond
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom.
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17
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Mantsiou A, Makridakis M, Fasoulakis K, Katafigiotis I, Constantinides CA, Zoidakis J, Roubelakis MG, Vlahou A, Lygirou V. Proteomics Analysis of Formalin Fixed Paraffin Embedded Tissues in the Investigation of Prostate Cancer. J Proteome Res 2019; 19:2631-2642. [PMID: 31682457 DOI: 10.1021/acs.jproteome.9b00587] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Prostate cancer (PCa) is one of the leading causes of death in men worldwide. The molecular features, associated with the onset and progression of the disease, are under vigorous investigation. Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources for large-scale studies; however, their application in proteomics is limited due to protein cross-linking. In this study, the adjustment of a protocol for the proteomic analysis of FFPE tissues was performed which was followed by a pilot application on FFPE PCa clinical samples to investigate whether the optimized protocol can provide biologically relevant data for the investigation of PCa. For the optimization, FFPE mouse tissues were processed using seven protein extraction protocols including combinations of homogenization methods (beads, sonication, boiling) and buffers (SDS based and urea-thiourea based). The proteome extraction efficacy was then evaluated based on protein identifications and reproducibility using SDS electrophoresis and high resolution LC-MS/MS analysis. Comparison between the FFPE and matched fresh frozen (FF) tissues, using an optimized protocol involving protein extraction with an SDS-based buffer following beads homogenization and boiling, showed a substantial overlap in protein identifications with a strong correlation in relative abundances (rs = 0.819, p < 0.001). Next, FFPE tissues (3 sections, 15 μm each per sample) from 10 patients with PCa corresponding to tumor (GS = 6 or GS ≥ 8) and adjacent benign regions were processed with the optimized protocol. Extracted proteins were analyzed by GeLC-MS/MS followed by statistical and bioinformatics analysis. Proteins significantly deregulated between PCa GS ≥ 8 and PCa GS = 6 represented extracellular matrix organization, gluconeogenesis, and phosphorylation pathways. Proteins deregulated between cancerous and adjacent benign tissues, reflected increased translation, peptide synthesis, and protein metabolism in the former, which is consistent with the literature. In conclusion, the results support the relevance of the proteomic findings in the context of PCa and the reliability of the optimized protocol for proteomics analysis of FFPE material.
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Affiliation(s)
- Anna Mantsiou
- Biomedical Research Foundation of the Academy of Athens, Biotechnology Division, 4 Soranou Ephessiou Street, Athens 11527, Greece
| | - Manousos Makridakis
- Biomedical Research Foundation of the Academy of Athens, Biotechnology Division, 4 Soranou Ephessiou Street, Athens 11527, Greece
| | - Konstantinos Fasoulakis
- Ippokrateio General Hospital of Athens, Department of Urology, 114 Vasilissis Sofias Avenue, Athens 11527, Greece
| | - Ioannis Katafigiotis
- National and Kapodistrian University of Athens, Medical School, 1st Urology Department, Laikon Hospital, 17 Agiou Thoma Street, Athens 11527, Greece
| | - Constantinos A Constantinides
- National and Kapodistrian University of Athens, Medical School, 1st Urology Department, Laikon Hospital, 17 Agiou Thoma Street, Athens 11527, Greece
| | - Jerome Zoidakis
- Biomedical Research Foundation of the Academy of Athens, Biotechnology Division, 4 Soranou Ephessiou Street, Athens 11527, Greece
| | - Maria G Roubelakis
- National and Kapodistrian University of Athens, Medical School, Laboratory of Biology, 75 Mikras Assias Street, Athens 11527, Greece
| | - Antonia Vlahou
- Biomedical Research Foundation of the Academy of Athens, Biotechnology Division, 4 Soranou Ephessiou Street, Athens 11527, Greece
| | - Vasiliki Lygirou
- Biomedical Research Foundation of the Academy of Athens, Biotechnology Division, 4 Soranou Ephessiou Street, Athens 11527, Greece
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18
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Zhu Y, Weiss T, Zhang Q, Sun R, Wang B, Yi X, Wu Z, Gao H, Cai X, Ruan G, Zhu T, Xu C, Lou S, Yu X, Gillet L, Blattmann P, Saba K, Fankhauser CD, Schmid MB, Rutishauser D, Ljubicic J, Christiansen A, Fritz C, Rupp NJ, Poyet C, Rushing E, Weller M, Roth P, Haralambieva E, Hofer S, Chen C, Jochum W, Gao X, Teng X, Chen L, Zhong Q, Wild PJ, Aebersold R, Guo T. High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification. Mol Oncol 2019; 13:2305-2328. [PMID: 31495056 PMCID: PMC6822243 DOI: 10.1002/1878-0261.12570] [Citation(s) in RCA: 107] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Revised: 08/09/2019] [Accepted: 09/03/2019] [Indexed: 11/06/2022] Open
Abstract
Formalin‐fixed, paraffin‐embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here, we developed a pressure cycling technology (PCT)‐SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B‐cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh‐frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between 1 and 15 years in a biobank and show a high degree of the proteome pattern similarity between two types of histological regions in small FFPE samples, that is, punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of a certain degree of biological variations. Applying the method to two independent DLBCL cohorts, we identified myeloperoxidase, a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.
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Affiliation(s)
- Yi Zhu
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China.,Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Switzerland
| | - Tobias Weiss
- Department of Neurology and Brain Tumor Center, University Hospital Zurich, University of Zurich, Switzerland
| | - Qiushi Zhang
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Rui Sun
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Bo Wang
- Department of Pathology, The First Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou, China
| | - Xiao Yi
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Zhicheng Wu
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Huanhuan Gao
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Xue Cai
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Guan Ruan
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Tiansheng Zhu
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Chao Xu
- College of Mathematics and Informatics, Digital Fujian Institute of Big Data Security Technology, Fujian Normal University, Fuzhou, China
| | - Sai Lou
- Phase I Clinical Research Center, Zhejiang Provincial People's Hospital, Hangzhou, China
| | - Xiaoyan Yu
- Department of Pathology, The Second Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou, China
| | - Ludovic Gillet
- Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Switzerland
| | - Peter Blattmann
- Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Switzerland
| | - Karim Saba
- Department of Urology, University Hospital Zurich, University of Zurich, Switzerland
| | | | - Michael B Schmid
- Department of Urology, University Hospital Zurich, University of Zurich, Switzerland
| | - Dorothea Rutishauser
- Department of Pathology and Molecular Pathology, University Hospital Zurich, University of Zurich, Switzerland
| | - Jelena Ljubicic
- Department of Pathology and Molecular Pathology, University Hospital Zurich, University of Zurich, Switzerland
| | - Ailsa Christiansen
- Department of Pathology and Molecular Pathology, University Hospital Zurich, University of Zurich, Switzerland
| | - Christine Fritz
- Department of Pathology and Molecular Pathology, University Hospital Zurich, University of Zurich, Switzerland
| | - Niels J Rupp
- Department of Pathology and Molecular Pathology, University Hospital Zurich, University of Zurich, Switzerland
| | - Cedric Poyet
- Department of Urology, University Hospital Zurich, University of Zurich, Switzerland
| | - Elisabeth Rushing
- Department of Neuropathology, University Hospital Zurich, University of Zurich, Switzerland
| | - Michael Weller
- Department of Neurology and Brain Tumor Center, University Hospital Zurich, University of Zurich, Switzerland
| | - Patrick Roth
- Department of Neurology and Brain Tumor Center, University Hospital Zurich, University of Zurich, Switzerland
| | - Eugenia Haralambieva
- Department of Pathology and Molecular Pathology, University Hospital Zurich, University of Zurich, Switzerland
| | - Silvia Hofer
- Division of Medical Oncology, Lucerne Cantonal Hospital and Cancer Center, Switzerland
| | | | - Wolfram Jochum
- Institute of Pathology, Cantonal Hospital St. Gallen, Switzerland
| | - Xiaofei Gao
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Xiaodong Teng
- Department of Pathology, The First Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou, China
| | - Lirong Chen
- Department of Pathology, The Second Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou, China
| | - Qing Zhong
- Department of Pathology and Molecular Pathology, University Hospital Zurich, University of Zurich, Switzerland.,Children's Medical Research Institute, University of Sydney, Australia
| | - Peter J Wild
- Department of Pathology and Molecular Pathology, University Hospital Zurich, University of Zurich, Switzerland.,Dr. Senckenberg Institute of Pathology, University Hospital Frankfurt, Frankfurt am Main, Germany
| | - Ruedi Aebersold
- Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Switzerland.,Faculty of Science, University of Zurich, Switzerland
| | - Tiannan Guo
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China.,Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Switzerland
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19
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Dapic I, Baljeu-Neuman L, Uwugiaren N, Kers J, Goodlett DR, Corthals GL. Proteome analysis of tissues by mass spectrometry. MASS SPECTROMETRY REVIEWS 2019; 38:403-441. [PMID: 31390493 DOI: 10.1002/mas.21598] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Accepted: 06/17/2019] [Indexed: 06/10/2023]
Abstract
Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh-frozen and formalin-fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue-sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom-up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys-C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue-specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.
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Affiliation(s)
- Irena Dapic
- International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland
| | | | - Naomi Uwugiaren
- International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland
| | - Jesper Kers
- Department of Pathology, Amsterdam Infection & Immunity Institute (AI&II), Amsterdam Cardiovascular Sciences (ACS), Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
- van 't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands
- Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA
| | - David R Goodlett
- International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland
- University of Maryland, 20N. Pine Street, Baltimore, MD 21201
| | - Garry L Corthals
- van 't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands
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20
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Contreras A, Leroy B, Mariage PA, Wattiez R. Proteomic analysis reveals novel insights into tanshinones biosynthesis in Salvia miltiorrhiza hairy roots. Sci Rep 2019; 9:5768. [PMID: 30962498 PMCID: PMC6453882 DOI: 10.1038/s41598-019-42164-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Accepted: 03/26/2019] [Indexed: 12/29/2022] Open
Abstract
Salvia miltiorrhiza is a medicinal plant highly appreciated by its content of tanshinones and salvianolic acids. Tanshinones are of particular relevance for their anti-oxidant, anti-tumoral and anti-inflammatory properties. Abiotic and biotic agents as silver nitrate and yeast extract have shown efficiently to stimulate tanshinone accumulation, but the underlying molecular mechanism remains essentially unknown. By using hairy roots as experimental material and the elicitors mentioned, were obtained up to 22 mg of tanshinones per gram of dry weight. Differential label-free quantitative proteomic analysis was applied to study the proteins involved in tanshinone biosynthesis. A total of 2650 proteins were identified in roots extracts, of which 893 showed statistically (p < 0.05) significant change in relative abundance compared to control roots, 251 proteins were upregulated and 642 downregulated. Among the upregulated proteins the predominant functional categories were metabolism (47%), stress defense (18%) and redox homeostasis (10%). Within the metabolism category, isoprenoid metabolism enzymes, cytochromes P450 and FAD-binding berberine proteins showed abundance profile linked to tanshinone concentration. The results presented here allowed to propose 5 new cytochromes P450 and 5 berberine enzymes as candidates to be involved into tanshinone biosynthesis, a novel finding that opens new avenues to improve tanshinone production through biotechnological approaches.
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Affiliation(s)
- Angela Contreras
- Proteomics and Microbiology department, Research center for Biosciences, University of Mons, 20 place du Parc, Mons, 7000, Belgium
| | - Baptiste Leroy
- Proteomics and Microbiology department, Research center for Biosciences, University of Mons, 20 place du Parc, Mons, 7000, Belgium
| | | | - Ruddy Wattiez
- Proteomics and Microbiology department, Research center for Biosciences, University of Mons, 20 place du Parc, Mons, 7000, Belgium.
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21
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Yun BH, Guo J, Turesky RJ. Formalin-Fixed Paraffin-Embedded Tissues-An Untapped Biospecimen for Biomonitoring DNA Adducts by Mass Spectrometry. TOXICS 2018; 6:E30. [PMID: 29865161 PMCID: PMC6027047 DOI: 10.3390/toxics6020030] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/28/2018] [Revised: 05/21/2018] [Accepted: 05/25/2018] [Indexed: 01/03/2023]
Abstract
The measurement of DNA adducts provides important information about human exposure to genotoxic chemicals and can be employed to elucidate mechanisms of DNA damage and repair. DNA adducts can serve as biomarkers for interspecies comparisons of the biologically effective dose of procarcinogens and permit extrapolation of genotoxicity data from animal studies for human risk assessment. One major challenge in DNA adduct biomarker research is the paucity of fresh frozen biopsy samples available for study. However, archived formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis of disease are often available. We have established robust methods to recover DNA free of crosslinks from FFPE tissues under mild conditions which permit quantitative measurements of DNA adducts by liquid chromatography-mass spectrometry. The technology is versatile and can be employed to screen for DNA adducts formed with a wide range of environmental and dietary carcinogens, some of which were retrieved from section-cuts of FFPE blocks stored at ambient temperature for up to nine years. The ability to retrospectively analyze FFPE tissues for DNA adducts for which there is clinical diagnosis of disease opens a previously untapped source of biospecimens for molecular epidemiology studies that seek to assess the causal role of environmental chemicals in cancer etiology.
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Affiliation(s)
- Byeong Hwa Yun
- Masonic Cancer Center and Department of Medicinal Chemistry, University of Minnesota, 2231 6th St. SE, Minneapolis, MN 55455, USA.
| | - Jingshu Guo
- Masonic Cancer Center and Department of Medicinal Chemistry, University of Minnesota, 2231 6th St. SE, Minneapolis, MN 55455, USA.
| | - Robert J Turesky
- Masonic Cancer Center and Department of Medicinal Chemistry, University of Minnesota, 2231 6th St. SE, Minneapolis, MN 55455, USA.
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22
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Biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: The Holy Grail for molecular diagnostics. J Pharm Biomed Anal 2018; 155:125-134. [PMID: 29627729 DOI: 10.1016/j.jpba.2018.03.065] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2018] [Revised: 03/30/2018] [Accepted: 03/31/2018] [Indexed: 02/07/2023]
Abstract
More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its proper architectural conservation of tissue structures and cellular shape. The huge collection of formalin-fixed, paraffin-embedded (FFPE) sample archives worldwide holds a large amount of unearthed information about diseases that could be the Holy Grail in contemporary biomarker research utilizing analytical omics based molecular diagnostics. The aim of this review is to critically evaluate the omics options for FFPE tissue sample analysis in the molecular diagnostics field.
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23
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Ostasiewicz P, Wiśniewski J. A Protocol for Large-Scale Proteomic Analysis of Microdissected Formalin Fixed and Paraffin Embedded Tissue. Methods Enzymol 2017; 585:159-176. [DOI: 10.1016/bs.mie.2016.09.017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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24
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Broeckx V, Boonen K, Pringels L, Sagaert X, Prenen H, Landuyt B, Schoofs L, Maes E. Comparison of multiple protein extraction buffers for GeLC-MS/MS proteomic analysis of liver and colon formalin-fixed, paraffin-embedded tissues. MOLECULAR BIOSYSTEMS 2016; 12:553-65. [PMID: 26676081 DOI: 10.1039/c5mb00670h] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a potential valuable source of samples for clinical research. Since these specimens are banked in hospital archives, large cohorts of samples can be collected in short periods of time which can all be linked with a patients' clinical history. Therefore, the use of FFPE tissue in protein biomarker discovery studies gains interest. However, despite the growing number of FFPE proteome studies in the literature, there is a lack of a FFPE proteomics standard operating procedure (SOP). One of the challenging steps in the development of such a SOP is the ability to obtain an efficient and repeatable extraction of full length FFPE proteins. In this study, the protein extraction efficiency of eight protein extraction buffers is critically compared with GeLC-MS/MS (1D gel electrophoresis followed by in-gel digestion and LC-MS/MS). The data variation caused by using these extraction buffers was investigated since the variation is a very important aspect when using FFPE tissue as a source for biomarker detection. In addition, a qualitative comparison was made between the protein extraction efficiency and repeatability for FFPE tissue and fresh frozen tissue.
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Affiliation(s)
- Valérie Broeckx
- Research Group of Functional Genomics and Proteomics, Department of Biology, University of Leuven, Zoological Institute, Naamsestraat 59, 3000 Leuven, Belgium.
| | - Kurt Boonen
- Research Group of Functional Genomics and Proteomics, Department of Biology, University of Leuven, Zoological Institute, Naamsestraat 59, 3000 Leuven, Belgium.
| | - Lentel Pringels
- Research Group of Functional Genomics and Proteomics, Department of Biology, University of Leuven, Zoological Institute, Naamsestraat 59, 3000 Leuven, Belgium.
| | - Xavier Sagaert
- Centre for Translational Cell and Tissue Research, University Hospital of Leuven, Herestraat 49, 3000 Leuven, Belgium
| | - Hans Prenen
- Department of Gastro-Enterology, Digestive Oncology Unit, University Hospital of Leuven, Herestraat 49, 3000 Leuven, Belgium
| | - Bart Landuyt
- Research Group of Functional Genomics and Proteomics, Department of Biology, University of Leuven, Zoological Institute, Naamsestraat 59, 3000 Leuven, Belgium.
| | - Liliane Schoofs
- Research Group of Functional Genomics and Proteomics, Department of Biology, University of Leuven, Zoological Institute, Naamsestraat 59, 3000 Leuven, Belgium.
| | - Evelyne Maes
- Flemish Institute for Technological Research (VITO), Boeretang 200, 2400 Mol, Belgium and Centre for Proteomics, University of Antwerp/Flemish Institute for Technological Research (VITO), Groenenborgerlaan 171, 2020 Antwerp, Belgium
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25
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Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations. Sci Rep 2016; 6:33999. [PMID: 27666089 PMCID: PMC5036045 DOI: 10.1038/srep33999] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Accepted: 09/01/2016] [Indexed: 12/14/2022] Open
Abstract
Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.
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26
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Yamamoto T, Kudo M, Peng WX, Takata H, Takakura H, Teduka K, Fujii T, Mitamura K, Taga A, Uchida E, Naito Z. Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue. Tumour Biol 2016; 37:13595-13606. [PMID: 27468721 PMCID: PMC5097088 DOI: 10.1007/s13277-016-5275-8] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2016] [Accepted: 07/15/2016] [Indexed: 12/12/2022] Open
Abstract
Colorectal cancer (CRC) is one of the most common cancers worldwide, and many patients are already at an advanced stage when they are diagnosed. Therefore, novel biomarkers for early detection of colorectal cancer are required. In this study, we performed a global shotgun proteomic analysis using formalin-fixed and paraffin-embedded (FFPE) CRC tissue. We identified 84 candidate proteins whose expression levels were differentially expressed in cancer and non-cancer regions. A label-free semiquantitative method based on spectral counting and gene ontology (GO) analysis led to a total of 21 candidate proteins that could potentially be detected in blood. Validation studies revealed cyclophilin A, annexin A2, and aldolase A mRNA and protein expression levels were significantly higher in cancer regions than in non-cancer regions. Moreover, an in vitro study showed that secretion of aldolase A into the culture medium was clearly suppressed in CRC cells compared to normal colon epithelium. These findings suggest that decreased aldolase A in blood may be a novel biomarker for the early detection of CRC.
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Affiliation(s)
- Tetsushi Yamamoto
- Pathological and Biomolecule Analyses Laboratory, Faculty of Pharmacy, Kindai University, Osaka, Japan
| | - Mitsuhiro Kudo
- Department of Integrated Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan
| | - Wei-Xia Peng
- Department of Integrated Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan
| | - Hideyuki Takata
- Department of Integrated Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan.,Departments of Gastrointestinal Hepato Biliary Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Hideki Takakura
- Pathological and Biomolecule Analyses Laboratory, Faculty of Pharmacy, Kindai University, Osaka, Japan
| | - Kiyoshi Teduka
- Department of Integrated Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan
| | - Takenori Fujii
- Department of Integrated Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan
| | - Kuniko Mitamura
- Pathological and Biomolecule Analyses Laboratory, Faculty of Pharmacy, Kindai University, Osaka, Japan
| | - Atsushi Taga
- Pathological and Biomolecule Analyses Laboratory, Faculty of Pharmacy, Kindai University, Osaka, Japan
| | - Eiji Uchida
- Departments of Gastrointestinal Hepato Biliary Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Zenya Naito
- Department of Integrated Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan.
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27
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Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval. Biotechniques 2016; 60:229-38. [DOI: 10.2144/000114414] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2015] [Accepted: 02/20/2016] [Indexed: 11/23/2022] Open
Abstract
Since emerging in the late 19th century, formaldehyde fixation has become a standard method for preservation of tissues from clinical samples. The advantage of formaldehyde fixation is that fixed tissues can be stored at room temperature for decades without concern for degradation. This has led to the generation of huge tissue banks containing thousands of clinically significant samples. Here we review techniques for proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue samples with a specific focus on the methods used to extract and break formaldehyde crosslinks. We also discuss an error-of-interpretation associated with the technique known as “antigen retrieval.” We have discovered that this term has been mistakenly applied to two disparate molecular techniques; therefore, we argue that a terminology change is needed to ensure accurate reporting of experimental results. Finally, we suggest that more investigation is required to fully understand the process of formaldehyde fixation and its subsequent reversal.
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28
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Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer's disease brain tissue. Sci Rep 2015; 5:15456. [PMID: 26487484 PMCID: PMC4614382 DOI: 10.1038/srep15456] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2015] [Accepted: 09/25/2015] [Indexed: 12/22/2022] Open
Abstract
The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer’s disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer’s disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.
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29
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Zhou Z, Irudayaraj J. A native chromatin extraction method based on salicylic acid coated magnetic nanoparticles and characterization of chromatin. Analyst 2015; 140:938-44. [PMID: 25475154 DOI: 10.1039/c4an01897d] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Native chromatin contains valuable genetic, epigenetic and structural information. Though DNA and nucleosome structures are well defined, less is known about the higher-order chromatin structure. Traditional chromatin extraction methods involve fixation, fragmentation and centrifugation, which might distort the higher-order structural information of native chromatin. We present a simple approach to isolate native chromatin from cultured mammalian cells using salicylic acid coated magnetic nanoparticles (SAMNPs). Chromatin is magnetically separated from cell lysates without any filtration or high-speed centrifugation. The purified chromatin is suitable for the examination of histone modifications and other chromatin associated proteins as confirmed by western blotting analysis. The structure of chromatin was determined by confocal fluorescence microscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM). High-resolution AFM and TEM images clearly show a classical bead-on-a-string structure. The higher-order chromatin structure is also determined via electron microscopy. Our method provides a simple, inexpensive and an environmentally friendly means to extract native chromatin not possible before, suitable for both biochemical and structural analysis.
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Affiliation(s)
- Zhongwu Zhou
- Bindley Bioscience Center and Birck Nanotechnology Center, Purdue University, West Lafayette, IN 47907, USA.
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30
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Weißer J, Lai ZW, Bronsert P, Kuehs M, Drendel V, Timme S, Kuesters S, Jilg CA, Wellner UF, Lassmann S, Werner M, Biniossek ML, Schilling O. Quantitative proteomic analysis of formalin-fixed, paraffin-embedded clear cell renal cell carcinoma tissue using stable isotopic dimethylation of primary amines. BMC Genomics 2015. [PMID: 26220445 PMCID: PMC4518706 DOI: 10.1186/s12864-015-1768-x] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Background Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most abundant resource of archived human specimens in pathology. Such tissue specimens are emerging as a highly valuable resource for translational proteomic studies. In quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness. Results In the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of FFPE specimens without interference from formalin employed in the FFPE process. Isotopic amine dimethylation of FFPE specimens showed equal labeling efficiency as for cryopreserved specimens. For both FFPE and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. In an initial application, we profiled proteome changes in clear cell renal cell carcinoma (ccRCC) FFPE tissue specimens compared to adjacent non–malignant renal tissue. Our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins. Conclusion Our study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of FFPE specimens and underlines proteome alterations in ccRCC. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1768-x) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- J Weißer
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany. .,Present address: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, A-1090, Vienna, Austria.
| | - Z W Lai
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
| | - P Bronsert
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany. .,Comprehensive Cancer Center Freiburg, Freiburg, Germany. .,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
| | - M Kuehs
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany.
| | - V Drendel
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany.
| | - S Timme
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany.
| | - S Kuesters
- Clinic for General and Visceral Surgery, University Medical Center Freiburg, Freiburg, Germany.
| | - C A Jilg
- Urologische Klinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Freiburg, 79106, Germany.
| | - U F Wellner
- Clinic for General and Visceral Surgery, University Medical Center Freiburg, Freiburg, Germany. .,Present address: Clinic for Surgery, University Clinic of Schleswig-Holstein Campus Lübeck, Lübeck, Germany.
| | - S Lassmann
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany. .,BIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79104, Freiburg, Germany. .,Comprehensive Cancer Center Freiburg, Freiburg, Germany. .,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
| | - M Werner
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany. .,Comprehensive Cancer Center Freiburg, Freiburg, Germany. .,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
| | - M L Biniossek
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
| | - O Schilling
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany. .,BIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79104, Freiburg, Germany. .,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
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31
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Chen Q, Yan G, Gao M, Zhang X. Ultrasensitive Proteome Profiling for 100 Living Cells by Direct Cell Injection, Online Digestion and Nano-LC-MS/MS Analysis. Anal Chem 2015; 87:6674-80. [PMID: 26061007 DOI: 10.1021/acs.analchem.5b00808] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future.
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Affiliation(s)
- Qi Chen
- Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
| | - Guoquan Yan
- Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
| | - Mingxia Gao
- Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
| | - Xiangmin Zhang
- Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
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32
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Takaya A, Peng WX, Ishino K, Kudo M, Yamamoto T, Wada R, Takeshita T, Naito Z. Cystatin B as a potential diagnostic biomarker in ovarian clear cell carcinoma. Int J Oncol 2015; 46:1573-81. [PMID: 25633807 DOI: 10.3892/ijo.2015.2858] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2014] [Accepted: 12/29/2014] [Indexed: 11/05/2022] Open
Abstract
Epithelial ovarian cancer (EOC) consists of four major subtypes: clear cell carcinoma (CCC), endometrioid adenocarcinoma (EA), mucinous adenocarcinoma (MA) and serous adenocarcinoma (SA). Relative to the other subtypes, the prognosis of CCC is poor due to a high recurrence rate and chemotherapy resistance, but CCC-specific biomarkers have yet to be identified. With the aim of identifying diagnostic and treatment biomarkers for CCC, we analyzed 96 cases of EOC (32 CCC, 13 EA, 19 MA, 32 SA) using liquid chromatography/mass spectrometry (LC/MS) followed by immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). Semi-quantification of protein differences between subtypes showed upregulation of 150 proteins and downregulation of 30 proteins in CCC relative to the other subtypes. Based on hierarchical clustering that revealed a marked distinction in the expression levels of cystatin B (CYTB) and Annexin A4 (ANXA4) in CCC relative to the other subtypes, we focused the study on CYTB and ANXA4 expression in EOCs by IHC, RT-qPCR and western blot analyses using tissue specimens and cultured cells. As a result, compared to the other subtypes, CCC showed significantly high expression levels of CYTB and ANXA4 in the analyses. To examine the possibility of CYTB and ANXA4 as serum diagnostic biomarkers of CCC, we checked the protein levels in conditioned media and cell lysates using culture cells. Compared with the other subtypes, CCC cell lines showed a significantly higher level of expression of CYTB in both conditioned media and cell lysates, while ANXA4 showed a higher level of expression in cell lysates only. Our results demonstrate that CYTB and ANXA4 overexpression may be related to carcinogenesis and histopathological differentiation of CCC. CYTB may be a secreted protein, and may serve as a potential serum diagnostic biomarker of CCC, while ANXA4 may be useful as an intracellular marker.
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Affiliation(s)
- Akane Takaya
- Department of Integrated Diagnostic Pathology, Nippon Medical School, Tokyo 113‑8602, Japan
| | - Wei-Xia Peng
- Department of Integrated Diagnostic Pathology, Nippon Medical School, Tokyo 113‑8602, Japan
| | - Kousuke Ishino
- Department of Integrated Diagnostic Pathology, Nippon Medical School, Tokyo 113‑8602, Japan
| | - Mitsuhiro Kudo
- Department of Integrated Diagnostic Pathology, Nippon Medical School, Tokyo 113‑8602, Japan
| | | | - Ryuichi Wada
- Department of Integrated Diagnostic Pathology, Nippon Medical School, Tokyo 113‑8602, Japan
| | - Toshiyuki Takeshita
- Division of Reproductive Medicine, Perinatology and Gynecologic Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo 113‑8603, Japan
| | - Zenya Naito
- Department of Integrated Diagnostic Pathology, Nippon Medical School, Tokyo 113‑8602, Japan
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Sullivan MA, Li S, Aroney STN, Deng B, Li C, Roura E, Schulz BL, Harcourt BE, Forbes JM, Gilbert RG. A rapid extraction method for glycogen from formalin-fixed liver. Carbohydr Polym 2014; 118:9-15. [PMID: 25542100 DOI: 10.1016/j.carbpol.2014.11.005] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2014] [Revised: 10/17/2014] [Accepted: 11/09/2014] [Indexed: 10/24/2022]
Abstract
Liver glycogen, a highly branched polymer, acts as our blood-glucose buffer. While past structural studies have extracted glycogen from fresh or frozen tissue using a cold-water, sucrose-gradient centrifugation technique, a method for the extraction of glycogen from formalin-fixed liver would allow the analysis of glycogen from human tissues that are routinely collected in pathology laboratories. In this study, both sucrose-gradient and formalin-fixed extraction techniques were carried out on piglet livers, with the yields, purities and size distributions (using size exclusion chromatography) compared. The formalin extraction technique, when combined with a protease treatment, resulted in higher yields (but lower purities) of glycogen with size distributions similar to the sucrose-gradient centrifugation technique. This formalin extraction procedure was also significantly faster, allowing glycogen extraction throughput to increase by an order of magnitude. Both extraction techniques were compatible with mass spectrometry proteomics, with analysis showing the two techniques were highly complementary.
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Affiliation(s)
- Mitchell A Sullivan
- Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China; The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD 4072, Australia.
| | - Shihan Li
- Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China; The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD 4072, Australia.
| | - Samuel T N Aroney
- The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD 4072, Australia.
| | - Bin Deng
- Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
| | - Cheng Li
- Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China; The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD 4072, Australia.
| | - Eugeni Roura
- The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD 4072, Australia.
| | - Benjamin L Schulz
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
| | - Brooke E Harcourt
- Glycation and Diabetes Complications, Mater Research-UQ, Translational Research Institute, Woolloongabba, QLD 4102, Australia.
| | - Josephine M Forbes
- Glycation and Diabetes Complications, Mater Research-UQ, Translational Research Institute, Woolloongabba, QLD 4102, Australia; Mater Clinical School, University of Queensland, Brisbane, QLD 4072, Australia.
| | - Robert G Gilbert
- Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China; The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD 4072, Australia.
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Assessment of the 2-d gel-based proteomics application of clinically archived formalin-fixed paraffin embedded tissues. Protein J 2014; 33:135-42. [PMID: 24500075 DOI: 10.1007/s10930-014-9545-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Hospital tissue repositories possess a vast and valuable supply of disease samples with matched retrospective clinical information. Detection and characterization of disease biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues will greatly aid the understanding of the diseases mechanisms and help in the development of diagnostic and prognostic markers. In this study, the possibility of using full-length proteins extracted from clinically archived FFPE tissues in two-dimensional (2-D) gel-based proteomics was evaluated. The evaluation was done based on two types of tumor tissues (breast and prostate) and two extraction protocols. The comparison of the 2-D patterns of FFPE extracts obtained by two extraction protocols with the matching frozen tissue extracts showed that only 7-10% of proteins from frozen tissues can be matched to proteins from FFPE tissues. Most of the spots in the 2-D FFPE's maps had pl 4-6, while the percentages of proteins with pl above 6 were 3-5 times lower in comparison to the fresh/frozen tissue. Despite the three-fold lower number of the detected spots in FFPE maps compared to matched fresh/frozen maps, 67-78% of protein spots in FFPE could not be matched to the corresponding spots in the fresh/frozen tissue maps indicating irreversible protein modifications. In conclusion, the inability to completely reverse the cross-linked complexes and overcome protein fragmentation with the present day FFPE extraction methods stands in the way of effective use of these samples in 2-D gel based proteomics studies.
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35
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Aichler M, Luber B, Lordick F, Walch A. Proteomic and metabolic prediction of response to therapy in gastric cancer. World J Gastroenterol 2014; 20:13648-13657. [PMID: 25320503 PMCID: PMC4194549 DOI: 10.3748/wjg.v20.i38.13648] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2013] [Accepted: 06/13/2014] [Indexed: 02/06/2023] Open
Abstract
Several new treatment options for gastric cancer have been introduced but the prognosis of patients diagnosed with gastric cancer is still poor. Disease prognosis could be improved for high-risk individuals by implementing earlier screenings. Because many patients are asymptomatic during the early stages of gastric cancer, the diagnosis is often delayed and patients present with unresectable locally advanced or metastatic disease. Cytotoxic treatment has been shown to prolong survival in general, but not all patients are responders. The application of targeted therapies and multimodal treatment has improved prognosis for those with advanced disease. However, these new therapeutic strategies do not uniformly benefit all patients. Predicting whether patients will respond to specific therapies would be of particular value and would allow for stratifying patients for personalized treatment strategies. Metabolic imaging by positron emission tomography was the first technique with the potential to predict the response of esophago-gastric cancer to neoadjuvant therapy. Exploring and validating tissue-based biomarkers are ongoing processes. In this review, we discuss the status of several targeted therapies for gastric cancer, as well as proteomic and metabolic methods for investigating biomarkers for therapy response prediction in gastric cancer.
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Gustafsson OJR, Arentz G, Hoffmann P. Proteomic developments in the analysis of formalin-fixed tissue. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2014; 1854:559-80. [PMID: 25315853 DOI: 10.1016/j.bbapap.2014.10.003] [Citation(s) in RCA: 82] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/14/2014] [Revised: 09/22/2014] [Accepted: 10/06/2014] [Indexed: 02/07/2023]
Abstract
Retrospective proteomic studies, including those which aim to elucidate the molecular mechanisms driving cancer, require the assembly and characterization of substantial patient tissue cohorts. The difficulty of maintaining and accessing native tissue archives has prompted the development of methods to access archives of formalin-fixed tissue. Formalin-fixed tissue archives, complete with patient meta data, have accumulated for decades, presenting an invaluable resource for these retrospective studies. This review presents the current knowledge concerning formalin-fixed tissue, with descriptions of the mechanisms of formalin fixation, protein extraction, top-down proteomics, bottom-up proteomics, quantitative proteomics, phospho- and glycoproteomics as well as imaging mass spectrometry. Particular attention has been given to the inclusion of proteomic investigations of archived tumour tissue. This article is part of a Special Issue entitled: Medical Proteomics.
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Affiliation(s)
- Ove J R Gustafsson
- Adelaide Proteomics Centre, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, Australia 5005
| | - Georgia Arentz
- Adelaide Proteomics Centre, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, Australia 5005
| | - Peter Hoffmann
- Adelaide Proteomics Centre, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, Australia 5005.
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Longuespée R, Fléron M, Pottier C, Quesada-Calvo F, Meuwis MA, Baiwir D, Smargiasso N, Mazzucchelli G, De Pauw-Gillet MC, Delvenne P, De Pauw E. Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology. OMICS-A JOURNAL OF INTEGRATIVE BIOLOGY 2014; 18:539-52. [DOI: 10.1089/omi.2014.0033] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- Rémi Longuespée
- Mass Spectrometry Laboratory, GIGA-Research, Department of Chemistry, University of Liège, Liège, Belgium
| | - Maximilien Fléron
- Mammalian Cell Culture Laboratory, GIGA-Research, Department of Biomedical and Preclinical Sciences, University of Liège, Liège, Belgium
| | - Charles Pottier
- Laboratory of Experimental Pathology, GIGA-Cancer, Department of Pathology, University of Liège, Liège, Belgium
| | - Florence Quesada-Calvo
- Hepato-Gastroenterology and Digestive Oncology Department, Liège University Hospital, University of Liège, Liège, Belgium
| | - Marie-Alice Meuwis
- Hepato-Gastroenterology and Digestive Oncology Department, Liège University Hospital, University of Liège, Liège, Belgium
| | - Dominique Baiwir
- GIGA-R, GIGA Proteomic Facilities, University of Liège, Liège, Belgium
| | - Nicolas Smargiasso
- Mass Spectrometry Laboratory, GIGA-Research, Department of Chemistry, University of Liège, Liège, Belgium
| | - Gabriel Mazzucchelli
- Mass Spectrometry Laboratory, GIGA-Research, Department of Chemistry, University of Liège, Liège, Belgium
| | - Marie-Claire De Pauw-Gillet
- Mammalian Cell Culture Laboratory, GIGA-Research, Department of Biomedical and Preclinical Sciences, University of Liège, Liège, Belgium
| | - Philippe Delvenne
- Laboratory of Experimental Pathology, GIGA-Cancer, Department of Pathology, University of Liège, Liège, Belgium
| | - Edwin De Pauw
- Mass Spectrometry Laboratory, GIGA-Research, Department of Chemistry, University of Liège, Liège, Belgium
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Bronsert P, Weißer J, Biniossek ML, Kuehs M, Mayer B, Drendel V, Timme S, Shahinian H, Küsters S, Wellner UF, Lassmann S, Werner M, Schilling O. Impact of routinely employed procedures for tissue processing on the proteomic analysis of formalin-fixed paraffin-embedded tissue. Proteomics Clin Appl 2014; 8:796-804. [PMID: 24888792 DOI: 10.1002/prca.201300082] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2013] [Revised: 05/08/2014] [Accepted: 05/26/2014] [Indexed: 12/20/2022]
Abstract
PURPOSE FFPE (formalin fixed, paraffin embedded) tissue cohorts represent an enduring archive of clinical specimens. Proteomic analysis of FFPE tissues is gaining interest for the in-depth analysis of aberrant proteome composition. Procedures for FFPE tissue processing are standardized but there is diversity regarding the different processing systems. This work focuses on three different processing methods commonly used in large European pathology institutes. EXPERIMENTAL DESIGN Formalin fixed tissue specimens of different tumors were serially sliced and processed with three different processing systems (xylene, ethanol/vacuum or microwave based). After paraffin embedding, they were subjected to MS-based proteomic analysis to investigate the impact of tissue processing techniques on the quality of proteomic analysis. Results were compared with proteomic analysis of corresponding cryopreserved tissue specimens. RESULTS All processing techniques achieved very good proteome coverage similar to the cryopreserved counterpart. Gene ontology profiles, relative protein abundances, and peptide modifications such as methionine oxidation or proteolytic truncation were highly similar for all techniques as well as for the cryopreserved samples. CONCLUSIONS AND CLINICAL RELEVANCE The results show that different processing procedures do not impede proteomic analysis as a robust and powerful approach for the identification of protein determinants and markers of disease processes and highlights the general robustness of FFPE-tissue based proteomics.
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Affiliation(s)
- Peter Bronsert
- Institute of Pathology, University Medical Center, Freiburg, Germany; Comprehensive Cancer Center, Freiburg, Germany
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Accessing microenvironment compartments in formalin-fixed paraffin-embedded tissues by protein expression analysis. Bioanalysis 2014; 5:2647-59. [PMID: 24180505 DOI: 10.4155/bio.13.222] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND Formalin-fixed paraffin-embedded (FFPE) samples are an outstanding source of new information regarding disease evolvements. Current research on new biomarkers and diseases features has recently invested resources in FFPE-related projects. RESULTS In order to initiate clinical protein-expression studies using minute amount of biological material, a workflow based on the combination of filter-assisted sample preparation with MS analysis and label-free quantification was developed. Xenograft lung tumor tissue was investigated as a model system. The workflow was optimized and characterized in terms of its reproducibility from a quantitative and qualitative point of view. We proposed a modification of the original filter-assisted sample preparation protocol to improve reproducibility and highlight its potential for the investigation of hydrophobic proteins. CONCLUSIONS Altogether the presented workflow allows analysis of FFPE samples with improvements in the analytical time and performance, and we show its application for lung cancer xenograft tissue samples.
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Fowler CB, O'Leary TJ, Mason JT. Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue. Expert Rev Proteomics 2014; 10:389-400. [PMID: 23992421 DOI: 10.1586/14789450.2013.820531] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.
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Affiliation(s)
- Carol B Fowler
- Laboratory of Proteomics and Protein Science, Washington DC Veterans Affairs Medical Center, Washington, DC, USA.
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41
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Rezaul K, Wilson LL, Han DK. Direct tissue proteomics in human diseases: potential applications to melanoma research. Expert Rev Proteomics 2014; 5:405-12. [DOI: 10.1586/14789450.5.3.405] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Giusti L, Lucacchini A. Proteomic studies of formalin-fixed paraffin-embedded tissues. Expert Rev Proteomics 2013; 10:165-77. [PMID: 23573783 DOI: 10.1586/epr.13.3] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable informational resource of histologically characterized specimens for proteomic studies. In this article, the authors review the advancement performed in the field of FFPE proteomics focusing on formaldehyde treatment and on strategies addressed to obtain the best recovery in the protein/peptide extraction. A variety of approaches have been used to characterize protein tissue extracts, and many efforts have been performed demonstrating the comparability between fresh/frozen and FFPE proteomes. Finally, the authors report and discuss the large numbers of works aimed at developing new strategies and sophisticated platforms in the analysis of FFPE samples to validate known potential biomarkers and to discover new ones.
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Affiliation(s)
- Laura Giusti
- Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
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43
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Kojima K, Bowersock GJ, Kojima C, Klug CA, Grizzle WE, Mobley JA. Validation of a robust proteomic analysis carried out on formalin-fixed paraffin-embedded tissues of the pancreas obtained from mouse and human. Proteomics 2013; 12:3393-402. [PMID: 22997103 DOI: 10.1002/pmic.201100663] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2011] [Revised: 08/17/2012] [Accepted: 09/05/2012] [Indexed: 01/02/2023]
Abstract
A number of reports have recently emerged with focus on extraction of proteins from formalin-fixed paraffin-embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D-nanoLC-MS(MS)(2) following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease.
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Affiliation(s)
- Kyoko Kojima
- Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA
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44
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Analysis of the formalin-fixed paraffin-embedded tissue proteome: pitfalls, challenges, and future prospectives. Amino Acids 2013; 45:205-18. [PMID: 23592010 DOI: 10.1007/s00726-013-1494-0] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2013] [Accepted: 04/02/2013] [Indexed: 10/26/2022]
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissues are a real treasure for retrospective analysis considering the amount of samples present in hospital archives, combined with pathological, clinical, and outcome information available for every sample. Although unlocking the proteome of these tissues is still a challenge, new approaches are being developed. In this review, we summarize the different mass spectrometry platforms that are used in human clinical studies to unravel the FFPE proteome. The different ways of extracting crosslinked proteins and the analytical strategies are pointed out. Also, the pitfalls and challenges concerning the quality of FFPE proteomic approaches are depicted. We also evaluated the potential of these analytical methods for future clinical FFPE proteomics applications.
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45
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Shi SR, Taylor CR, Fowler CB, Mason JT. Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies. Proteomics Clin Appl 2013; 7:264-72. [PMID: 23339100 DOI: 10.1002/prca.201200031] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2012] [Revised: 10/12/2012] [Accepted: 11/06/2012] [Indexed: 02/01/2023]
Abstract
Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.
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Affiliation(s)
- Shan-Rong Shi
- Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
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46
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Wiśniewski JR, Duś K, Mann M. Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins. Proteomics Clin Appl 2013; 7:225-33. [PMID: 23090905 DOI: 10.1002/prca.201200046] [Citation(s) in RCA: 113] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Revised: 08/03/2012] [Accepted: 09/28/2012] [Indexed: 02/06/2023]
Abstract
PURPOSE Archival formalin-fixed and paraffin-embedded clinical samples represent a very diverse source of material for proteomic investigation of diseases, often with follow-up patient information. Here, we describe an analytical workflow for analysis of laser-capture microdissected formalin-fixed and paraffin-embedded samples that allows studying proteomes to a depth of 10 000 proteins per sample. EXPERIMENTAL DESIGN The workflow involves lysis of tissue in SDS-containing buffer, detergent removal, and consecutive digestion of the proteins with two enzymes by the multienzyme digestion filter-aided sample preparation method. Resulting peptides are fractionated by pipette-tip based strong anion exchange into six fractions and analyzed by LC-MS/MS on a bench top quadrupole Orbitrap mass spectrometer. RESULTS Analysis of the data using the MaxQuant software resulted in the identification of 9502 ± 28 protein groups per a 110 nL sample of microdissected cells from human colonic adenoma. This depth of proteome analysis enables systemic insights into the organization of the adenoma cells and an estimation of the abundances of known biomarkers. It also allows the identification of proteins expressed from tumor suppressors, oncogenes, and other key players in the development and progression of the colorectal cancer. CONCLUSION AND CLINICAL RELEVANCE Our proteomic platform can be used for quantitative comparisons between samples representing different stages of diseases and thus can be applied to the discovery of biomarkers or drug targets.
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Affiliation(s)
- Jacek R Wiśniewski
- Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Martinsried, Germany.
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47
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Gámez-Pozo A, Ferrer NI, Ciruelos E, López-Vacas R, Martínez FG, Espinosa E, Vara JÁF. Shotgun proteomics of archival triple-negative breast cancer samples. Proteomics Clin Appl 2013; 7:283-91. [DOI: 10.1002/prca.201200048] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Revised: 11/15/2012] [Accepted: 12/10/2012] [Indexed: 11/07/2022]
Affiliation(s)
- Angelo Gámez-Pozo
- Laboratorio de Oncología y Patología Molecular, Instituto de Genética Médica y Molecular-INGEMM; Instituto de Investigación Hospital Universitario La Paz-IdiPAZ; Madrid; Spain
| | - Nuria Ibarz Ferrer
- Unidad de Proteómica; Centro Nacional de Investigaciones Oncológicas (CNIO); Madrid; Spain
| | - Eva Ciruelos
- Servicio de Oncología; Instituto de Investigación Hospital Universitario Doce de Octubre-i+12; Madrid; Spain
| | - Rocío López-Vacas
- Laboratorio de Oncología y Patología Molecular, Instituto de Genética Médica y Molecular-INGEMM; Instituto de Investigación Hospital Universitario La Paz-IdiPAZ; Madrid; Spain
| | | | - Enrique Espinosa
- Servicio de Oncología Médica; Instituto de Investigación Hospital Universitario La Paz-IdiPAZ; Madrid; Spain
| | - Juan Ángel Fresno Vara
- Laboratorio de Oncología y Patología Molecular, Instituto de Genética Médica y Molecular-INGEMM; Instituto de Investigación Hospital Universitario La Paz-IdiPAZ; Madrid; Spain
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Thasler WE, Thasler RMK, Schelcher C, Jauch KW. Biobanking for research in surgery: are surgeons in charge for advancing translational research or mere assistants in biomaterial and data preservation? Langenbecks Arch Surg 2013; 398:487-99. [PMID: 23430290 DOI: 10.1007/s00423-013-1060-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2013] [Accepted: 01/31/2013] [Indexed: 01/09/2023]
Abstract
BACKGROUND High-quality biospecimens of human origin with annotated clinical and procedural data are an important tool for biomedical research, not only to map physiology, pathophysiology and aetiology but also to go beyond in translational research. This has opened a new special field of research known as 'biobanking', which focuses on how to collect, store and provide these specimens and data, and which is substantially supported by national and European funding. PURPOSE An overview on biobanking is given, with a closer look on a clinical setting, concerning a necessary distinction from clinical trials and studies as well as a comparison of prospective sample collection with secondary use of archived samples from diagnostics. Based on a summary of possible use and scientific impact of human tissue in research, it is shown how surgical expertise boosts the scientific value of specimens and data. Finally, an assessment of legal and ethical issues especially from a surgical perspective is given, followed by a model of interdisciplinary biobanking within a joint 'centre' that as synergistic structure merges essential input from surgery as well as laboratory medicine, pathology and biometry. CONCLUSION Within the domain of biobanking, surgeons have to develop a better awareness of their role within translational research, not only on the level of medical faculties but also as nationally and internationally funded initiatives. Therefore, the authors suggest a platform for biobanking within the German association of surgeons in analogy to the existing special interest group for clinical trials.
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Affiliation(s)
- Wolfgang E Thasler
- Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Ludwig Maximilians University, Marchioninistr. 15, 81377 Munich, Germany.
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Zhang Y, Hu Z, Qin H, Wei X, Cheng K, Liu F, Wu R, Zou H. Highly Efficient Extraction of Cellular Nucleic Acid Associated Proteins in Vitro with Magnetic Oxidized Carbon Nanotubes. Anal Chem 2012; 84:10454-62. [DOI: 10.1021/ac302695u] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Affiliation(s)
- Yi Zhang
- CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
- Graduate School of Chinese Academy of Sciences, Beijing 100049, China
| | - Zhengyan Hu
- CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
- Graduate School of Chinese Academy of Sciences, Beijing 100049, China
| | - Hongqiang Qin
- CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
- Graduate School of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiaoluan Wei
- CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
| | - Kai Cheng
- CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
- Graduate School of Chinese Academy of Sciences, Beijing 100049, China
| | - Fangjie Liu
- CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
- Graduate School of Chinese Academy of Sciences, Beijing 100049, China
| | - Ren’an Wu
- CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
| | - Hanfa Zou
- CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
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Comparability of differential proteomics data generated from paired archival fresh-frozen and formalin-fixed samples by GeLC-MS/MS and spectral counting. J Proteomics 2012; 77:561-76. [PMID: 23043969 DOI: 10.1016/j.jprot.2012.09.033] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2012] [Revised: 09/17/2012] [Accepted: 09/22/2012] [Indexed: 11/22/2022]
Abstract
In this study, a Veterinary Department repository composed by paired formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FrFr) sets of the same tissues, routinely archived in the typical conditions of a clinical setting, was exploited to perform a comparative evaluation of the results generated by GeLC-MS/MS (1-DE followed by in-gel digestion and LC-MS/MS) and spectral counting with the two types of archival samples. Therefore, two parallel differential proteomic studies were performed using 3 canine mammary carcinomas and 3 normal controls in a paired fashion (6 FrFr and 6 FFPE in total). As a result, the FrFr and FFPE differential proteomic datasets exhibited fair consistency in differential expression trends, according to protein molecular function, cellular localization, networks, and pathways. However, FFPE samples were globally slightly less informative, especially concerning the high-MW subproteome. As a further investigation, new insights into the molecular aspects of protein fixation and retrieval were obtained. In conclusion, archival FFPE samples can be reliably used for differential proteomics studies employing a spectral counting GeLC-MS/MS approach, although some typical biases need to be taken into account, and FrFr specimens (when available) should still be considered as the gold standard for clinical proteomics.
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