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Bjelosevic S, Pascovici D, Ping H, Karlaftis V, Zaw T, Song X, Molloy MP, Monagle P, Ignjatovic V. Quantitative Age-specific Variability of Plasma Proteins in Healthy Neonates, Children and Adults. Mol Cell Proteomics 2017; 16:924-935. [PMID: 28336724 DOI: 10.1074/mcp.m116.066720] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2017] [Revised: 02/26/2017] [Indexed: 02/02/2023] Open
Abstract
Human blood plasma is a complex biological fluid containing soluble proteins, sugars, hormones, electrolytes, and dissolved gasses. As plasma interacts with a wide array of bodily systems, changes in protein expression, or the presence or absence of specific proteins are regularly used in the clinic as a molecular biomarker tool. A large body of literature exists detailing proteomic changes in pathologic contexts, however little research has been conducted on the quantitation of the plasma proteome in age-specific, healthy subjects, especially in pediatrics. In this study, we utilized SWATH-MS to identify and quantify proteins in the blood plasma of healthy neonates, infants under 1 year of age, children between 1-5 years, and adults. We identified more than 100 proteins that showed significant differential expression levels across these age groups, and we analyzed variation in protein expression across the age spectrum. The plasma proteomic profiles of neonates were strikingly dissimilar to the older children and adults. By extracting the SWATH data against a large human spectral library we increased protein identification more than 6-fold (940 proteins) and confirmed the concentrations of several of these using ELISA. The results of this study map the variation in expression of proteins and pathways often implicated in disease, and so have significant clinical implication.
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Affiliation(s)
- Stefan Bjelosevic
- From the ‡Hematology Research Laboratory, Murdoch Childrens Research Institute, Melbourne, VIC 3052, Australia.,§Department of Paediatrics, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Dana Pascovici
- ¶Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW 2113, Australia
| | - Hui Ping
- From the ‡Hematology Research Laboratory, Murdoch Childrens Research Institute, Melbourne, VIC 3052, Australia.,§Department of Paediatrics, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Vasiliki Karlaftis
- From the ‡Hematology Research Laboratory, Murdoch Childrens Research Institute, Melbourne, VIC 3052, Australia
| | - Thiri Zaw
- ¶Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW 2113, Australia
| | - Xiaomin Song
- ¶Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW 2113, Australia
| | - Mark P Molloy
- ¶Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW 2113, Australia
| | - Paul Monagle
- From the ‡Hematology Research Laboratory, Murdoch Childrens Research Institute, Melbourne, VIC 3052, Australia.,§Department of Paediatrics, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Vera Ignjatovic
- From the ‡Hematology Research Laboratory, Murdoch Childrens Research Institute, Melbourne, VIC 3052, Australia; .,§Department of Paediatrics, The University of Melbourne, Melbourne, VIC 3010, Australia
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Miah S, Banks CAS, Adams MK, Florens L, Lukong KE, Washburn MP. Advancement of mass spectrometry-based proteomics technologies to explore triple negative breast cancer. MOLECULAR BIOSYSTEMS 2016; 13:42-55. [PMID: 27891540 PMCID: PMC5173390 DOI: 10.1039/c6mb00639f] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Understanding the complexity of cancer biology requires extensive information about the cancer proteome over the course of the disease. The recent advances in mass spectrometry-based proteomics technologies have led to the accumulation of an incredible amount of such proteomic information. This information allows us to identify protein signatures or protein biomarkers, which can be used to improve cancer diagnosis, prognosis and treatment. For example, mass spectrometry-based proteomics has been used in breast cancer research for over two decades to elucidate protein function. Breast cancer is a heterogeneous group of diseases with distinct molecular features that are reflected in tumour characteristics and clinical outcomes. Compared with all other subtypes of breast cancer, triple-negative breast cancer is perhaps the most distinct in nature and heterogeneity. In this review, we provide an introductory overview of the application of advanced proteomic technologies to triple-negative breast cancer research.
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Affiliation(s)
- Sayem Miah
- Stowers Institute for Medical Research, 1000 E. 50th St, Kansas City, MO 64110, USA. and Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Charles A S Banks
- Stowers Institute for Medical Research, 1000 E. 50th St, Kansas City, MO 64110, USA.
| | - Mark K Adams
- Stowers Institute for Medical Research, 1000 E. 50th St, Kansas City, MO 64110, USA.
| | - Laurence Florens
- Stowers Institute for Medical Research, 1000 E. 50th St, Kansas City, MO 64110, USA.
| | - Kiven E Lukong
- Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Michael P Washburn
- Stowers Institute for Medical Research, 1000 E. 50th St, Kansas City, MO 64110, USA. and Departments of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA
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Vaiopoulou A, Gazouli M, Papadopoulou A, Anagnostopoulos AK, Karamanolis G, Theodoropoulos GE, M’Koma A, Tsangaris GT. Serum protein profiling of adults and children with Crohn disease. J Pediatr Gastroenterol Nutr 2015; 60:42-47. [PMID: 25250685 PMCID: PMC4276513 DOI: 10.1097/mpg.0000000000000579] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
OBJECTIVES Crohn disease (CD) and ulcerative colitis (UC), known collectively as inflammatory bowel diseases (IBDs), are chronic immunoinflammatory pathologies of unknown aetiology. Despite the frequent use of biomarkers in medical practice, there is a relative lack of information regarding validated paediatric biomarkers for IBD. Furthermore, biomarkers proved to be efficacious in adults are frequently extrapolated to the paediatric clinical setting without considering that the pathogenesis of many diseases is distinctly different in children. In the present study, proteomics technology was used to monitor differences in protein expression among adult and young patients with CD, identify a panel of candidate protein biomarkers that may be used to improve prognostic-diagnostic accuracy, and advance paediatric medical care. METHODS Male and female serum samples from 12 adults and 12 children with active CD were subjected to 2-dimensional gel electrophoresis. Following the relative quantitation of protein spots exhibiting a differential expression between the 2 groups by densitometry, the spots were further characterized by matrix-assisted laser desorption tandem time-of-flight mass spectrometer. The results were confirmed by Western blot analysis. RESULTS Clusterin was found to be significantly overexpressed in adults with CD, whereas ceruloplasmin and apolipoprotein B-100 were found to be significantly overexpressed in children, indicating that the expression of these proteins may be implicated in the onset or progression of CD in these 2 subgroups of patients. CONCLUSIONS Interestingly, we found a differential expression of several proteins in adults versus paediatric patients with CD. Undoubtedly, future experiments using a larger cohort of patients with CD are needed to evaluate the relevance of our preliminary findings.
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Affiliation(s)
- Anna Vaiopoulou
- Department of Basic Medical Sciences, Laboratory of Biology, School of Medicine, University of Athens, Greece
| | - Maria Gazouli
- Department of Basic Medical Sciences, Laboratory of Biology, School of Medicine, University of Athens, Greece
| | - Aggeliki Papadopoulou
- Proteomics Research Unit, Biomedical Research Foundation of the Academy of Athens (IIBEAA), Greece
| | | | - George Karamanolis
- Gastroenterology Unit, 2 Department of Surgery, “Aretaieio” University Hospital, Athens
| | - George E. Theodoropoulos
- First Propaedeutic Surgical Department, Hippocration University Hospital, University of Athens, Athens, Greece
| | - Amosy M’Koma
- Department of Biochemistry and Cancer Biology, Meharry Medical School of Medicine and Department of General Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, U.S.A
| | - George T. Tsangaris
- Proteomics Research Unit, Biomedical Research Foundation of the Academy of Athens (IIBEAA), Greece
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Kapoor I, Pal P, Lochab S, Kanaujiya JK, Trivedi AK. Proteomics approaches for myeloid leukemia drug discovery. Expert Opin Drug Discov 2012; 7:1165-75. [DOI: 10.1517/17460441.2012.724055] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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Tan HT, Lee YH, Chung MCM. Cancer proteomics. MASS SPECTROMETRY REVIEWS 2012; 31:583-605. [PMID: 22422534 DOI: 10.1002/mas.20356] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/29/2011] [Revised: 11/16/2011] [Accepted: 11/16/2011] [Indexed: 05/31/2023]
Abstract
Cancer presents high mortality and morbidity globally, largely due to its complex and heterogenous nature, and lack of biomarkers for early diagnosis. A proteomics study of cancer aims to identify and characterize functional proteins that drive the transformation of malignancy, and to discover biomarkers to detect early-stage cancer, predict prognosis, determine therapy efficacy, identify novel drug targets, and ultimately develop personalized medicine. The various sources of human samples such as cell lines, tissues, and plasma/serum are probed by a plethora of proteomics tools to discover novel biomarkers and elucidate mechanisms of tumorigenesis. Innovative proteomics technologies and strategies have been designed for protein identification, quantitation, fractionation, and enrichment to delve deeper into the oncoproteome. In addition, there is the need for high-throughput methods for biomarker validation, and integration of the various platforms of oncoproteome data to fully comprehend cancer biology.
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Affiliation(s)
- Hwee Tong Tan
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
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Kowdley G, Srikantan S, Abdelmohsen K, Gorospe M, Khan J. Molecular biology techniques for the surgeon. World J Surg Proced 2012; 2:5-15. [DOI: 10.5412/wjsp.v2.i2.5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
New technologies are constantly being introduced into the medical and surgical fields. These technologies come in the form of newer medicines, imaging methods and prognostic tools, among others, and allow clinicians to make more rational and informed decisions on the care of their patients. Many of these technologies utilize advanced techniques which are at the forefront of many research fields and represent a transition of bench advances into the clinical realm. This review will highlight four technologies that are at the forefront in the treatment of oncology patients treated by surgeons on a daily basis. Circulating tumor cells, microarray analysis, proteomic studies and rapid sequencing technologies will be highlighted. These technologies will be reviewed and their potential use in the care of surgical patients will be discussed.
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Ignjatovic V, Lai C, Summerhayes R, Mathesius U, Tawfilis S, Perugini MA, Monagle P. Age-related differences in plasma proteins: how plasma proteins change from neonates to adults. PLoS One 2011; 6:e17213. [PMID: 21365000 PMCID: PMC3041803 DOI: 10.1371/journal.pone.0017213] [Citation(s) in RCA: 98] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2010] [Accepted: 01/25/2011] [Indexed: 11/19/2022] Open
Abstract
The incidence of major diseases such as cardiovascular disease, thrombosis and cancer increases with age and is the major cause of mortality world-wide, with neonates and children somehow protected from such diseases of ageing. We hypothesized that there are major developmental differences in plasma proteins and that these contribute to age-related changes in the incidence of major diseases. We evaluated the human plasma proteome in healthy neonates, children and adults using the 2D-DIGE approach. We demonstrate significant changes in number and abundance of up to 100 protein spots that have marked differences in during the transition of the plasma proteome from neonate and child through to adult. These proteins are known to be involved in numerous physiological processes such as iron transport and homeostasis, immune response, haemostasis and apoptosis, amongst others. Importantly, we determined that the proteins that are differentially expressed with age are not the same proteins that are differentially expressed with gender and that the degree of phosphorylation of plasma proteins also changes with age. Given the multi-functionality of these proteins in human physiology, understanding the differences in the plasma proteome in neonates and children compared to adults will make a major contribution to our understanding of developmental biology in humans.
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Affiliation(s)
- Vera Ignjatovic
- Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
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8
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Hjelle SM, Forthun RB, Haaland I, Reikvam H, Sjøholt G, Bruserud O, Gjertsen BT. Clinical proteomics of myeloid leukemia. Genome Med 2010; 2:41. [PMID: 20587003 PMCID: PMC2905101 DOI: 10.1186/gm162] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Myeloid leukemias are a heterogeneous group of diseases originating from bone marrow myeloid progenitor cells. Patients with myeloid leukemias can achieve long-term survival through targeted therapy, cure after intensive chemotherapy or short-term survival because of highly chemoresistant disease. Therefore, despite the development of advanced molecular diagnostics, there is an unmet need for efficient therapy that reflects the advanced diagnostics. Although the molecular design of therapeutic agents is aimed at interacting with specific proteins identified through molecular diagnostics, the majority of therapeutic agents act on multiple protein targets. Ongoing studies on the leukemic cell proteome will probably identify a large number of new biomarkers, and the prediction of response to therapy through these markers is an interesting avenue for future personalized medicine. Mass spectrometric protein detection is a fundamental technique in clinical proteomics, and selected tools are presented, including stable isotope labeling with amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantification (iTRAQ) and multiple reaction monitoring (MRM), as well as single cell determination. We suggest that protein analysis will play not only a supplementary, but also a prominent role in future molecular diagnostics, and we outline how accurate knowledge of the molecular therapeutic targets can be used to monitor therapy response.
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Affiliation(s)
- Sigrun M Hjelle
- Institute of Medicine, Hematology Section, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway.
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Horvatovich P, Hoekman B, Govorukhina N, Bischoff R. Multidimensional chromatography coupled to mass spectrometry in analysing complex proteomics samples. J Sep Sci 2010; 33:1421-37. [DOI: 10.1002/jssc.201000050] [Citation(s) in RCA: 76] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Péter Horvatovich
- Analytical Biochemistry, Department of Pharmacy, University of Groningen, Groningen, The Netherlands
| | - Berend Hoekman
- Analytical Biochemistry, Department of Pharmacy, University of Groningen, Groningen, The Netherlands
| | - Natalia Govorukhina
- Analytical Biochemistry, Department of Pharmacy, University of Groningen, Groningen, The Netherlands
| | - Rainer Bischoff
- Analytical Biochemistry, Department of Pharmacy, University of Groningen, Groningen, The Netherlands
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Greco TM, Seeholzer SH, Mak A, Spruce L, Ischiropoulos H. Quantitative mass spectrometry-based proteomics reveals the dynamic range of primary mouse astrocyte protein secretion. J Proteome Res 2010; 9:2764-74. [PMID: 20329800 PMCID: PMC2866110 DOI: 10.1021/pr100134n] [Citation(s) in RCA: 91] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Growing appreciation for astrocytes as active participants in nervous system development, neurovascular metabolic coupling, and neurological disease progression has stimulated recent investigation into specific astrocyte-secreted proteins that may mediate these functions. The current work utilized SILAC-generated isotope reference proteomes to quantify relative protein abundances between the astrocyte proteome and secretome. Multidimensional GeLC-MS/MS analysis of astrocyte conditioned media and cell lysates resulted in the relative quantification of 516 proteins, 92 of which were greater than 1.5-fold enriched in astrocyte-conditioned media (ACM). Eighty of the ACM-enriched proteins had N-terminal signal peptides, comprising well-known classically secreted proteins, such as apolipoprotein E and SPARC, and several cathepsins that localize to endosomal/lysosomal compartments. The remaining twelve ACM-enriched proteins, such as vimentin, ferritins, and histones, lacked N-terminal signal peptides. Also, 47 proteins contained predicted N-terminal signal peptides but were not enriched in ACM (<1.5-fold), 25 of which were localized to ER, Golgi, or mitochondria membrane-bound compartments. Overall, by combining quantitative proteomics with subcellular localization prediction, an informative description of protein distribution can be obtained, providing insights into protein secretion.
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Affiliation(s)
- Todd M. Greco
- Department of Pediatrics, The Children’s Hospital of Philadelphia Research Institute and The University of Pennsylvania Philadelphia, PA, 19104, USA
| | - Steven H. Seeholzer
- Department of Pediatrics, The Children’s Hospital of Philadelphia Research Institute and The University of Pennsylvania Philadelphia, PA, 19104, USA
| | - Adrian Mak
- Department of Pediatrics, The Children’s Hospital of Philadelphia Research Institute and The University of Pennsylvania Philadelphia, PA, 19104, USA
| | - Lynn Spruce
- Department of Pediatrics, The Children’s Hospital of Philadelphia Research Institute and The University of Pennsylvania Philadelphia, PA, 19104, USA
| | - Harry Ischiropoulos
- Department of Pediatrics, The Children’s Hospital of Philadelphia Research Institute and The University of Pennsylvania Philadelphia, PA, 19104, USA
- Department of Pharmacology, The Children’s Hospital of Philadelphia Research Institute and The University of Pennsylvania Philadelphia, PA, 19104, USA
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Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin inhibits the proliferation of ARPE-19 cells. J Biomed Sci 2010; 17:30. [PMID: 20416086 PMCID: PMC2873497 DOI: 10.1186/1423-0127-17-30] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2009] [Accepted: 04/23/2010] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin) on human retinal pigment epithelial cells is investigated. METHODS MTT and flow cytometry were used to study the antiproliferative effects of the 17-AAG treatment of ARPE-19 cells. 2D gel electrophoresis (2-DE) and mass spectrometry were applied to detect the altered expression of proteins, which was verified by real-time PCR. Gene Ontology analysis and Ingenuity Pathway Analysis (IPA) were utilized to analyze the signaling pathways, cellular location, function, and network connections of the identified proteins. And SOD assay was employed to confirm the analysis. RESULTS 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis. Proteomic analysis revealed that the expression of 94 proteins was altered by a factor of more than 1.5 following exposure to 17-AAG. Of these 94, 87 proteins were identified. Real-time PCR results indicated that Hsp90 and Hsp70, which were not identified by proteomic analysis, were both upregulated upon 17-AAG treatment. IPA revealed that most of the proteins have functions that are related to oxidative stress, as verified by SOD assay, while canonical pathway analysis revealed glycolysis/gluconeogenesis. CONCLUSIONS 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis, and possibly by oxidative stress.
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12
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Xu X, Qiao M, Zhang Y, Jiang Y, Wei P, Yao J, Gu B, Wang Y, Lu J, Wang Z, Tang Z, Sun Y, Wu W, Shi Q. Quantitative proteomics study of breast cancer cell lines isolated from a single patient: Discovery of TIMM17A as a marker for breast cancer. Proteomics 2010; 10:1374-90. [DOI: 10.1002/pmic.200900380] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Abstract
The ability to conduct validated analyses of biomarkers is critically important in order to establish the sensitivity and selectivity of the biomarker in identifying a particular disease. The use of stable-isotope dilution (SID) methodology in combination with LC–MS/MS provides the highest possible analytical specificity for quantitative determinations. This methodology is now widely used in the discovery and validation of putative exposure and disease biomarkers. This review will describe the application of SID LC–MS methodology for the analysis of small-molecule and protein biomarkers. It will also discuss potential future directions for the use of this methodology for rigorous biomarker analysis.
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Affiliation(s)
- Eugene Ciccimaro
- Thermo Fisher Scientific, 265 Davidson Avenue, Somerset, NJ 08873–4120, USA
| | - Ian A Blair
- Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania School of Medicine, 421 Curie Blvd, Philadelphia, PA 19104–6160, USA
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Proteomic analysis of lymphoid and haematopoietic neoplasms: There's more than biomarker discovery. J Proteomics 2010; 73:508-20. [DOI: 10.1016/j.jprot.2009.08.012] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2009] [Revised: 08/26/2009] [Accepted: 08/27/2009] [Indexed: 12/29/2022]
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15
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Williams D, Ackloo S, Zhu P, Bowden P, Evans KR, Addison CL, Lock C, Marshall JG. Precipitation and selective extraction of human serum endogenous peptides with analysis by quadrupole time-of-flight mass spectrometry reveals posttranslational modifications and low-abundance peptides. Anal Bioanal Chem 2009; 396:1223-47. [PMID: 20033139 DOI: 10.1007/s00216-009-3345-0] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2009] [Revised: 11/23/2009] [Accepted: 11/24/2009] [Indexed: 11/30/2022]
Abstract
The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous blood peptides can be effectively enriched by precipitation of the serum with organic solvents followed by selective extraction of peptides using aqueous solutions modified with organic solvents. Polypeptides on filter paper were assayed with Coomasie brilliant blue binding. The polypeptides were resolved by detergent tricine polyacrylamide electrophoresis and visualized by diamine silver staining. Peptides in the extracts were collected by C18 and analyzed by matrix-assisted laser desorption/ionization and liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) quadrupole time-of-flight MS/MS. Peptides were resolved as multiple isotopic peaks in MS mode with mass deviation of 0.1 Da or less and similar accuracy for fragments. The sensitivity of MS and MS/MS analysis was estimated to be in the picomolar range or less. The peptide composition of the extracts was dependent on solvent formulation. Multiple peptides from apolipoproteins, complement proteins, coagulation factors, and many others were identified by X!Tandem with high mass accuracy of peptide ions and fragments from collision-induced dissociation. Many previously unreported posttranslational modifications of peptides including phosphorylations, oxidations, glycosylations, and others were detected with high mass accuracy and may be of clinical importance. About 4,630 redundant peptides were identified with 99% confidence separately, and together some 1,251 distinct proteins were identified with 99% confidence or greater using the Paragon algorithm.
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Affiliation(s)
- Declan Williams
- Department of Chemistry and Biology, Faculty of Engineering and Applied Science, Ryerson University, 350 Victoria Street, Toronto, ON, M5B 2K3, Canada
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Li H, He Y, Ding G, Wang C, Xie L, Li Y. dbDEPC: a database of differentially expressed proteins in human cancers. Nucleic Acids Res 2009; 38:D658-64. [PMID: 19900968 PMCID: PMC2808941 DOI: 10.1093/nar/gkp933] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Cancer-related investigations have long been in the limelight of biomedical research. Years of effort from scientists and doctors worldwide have generated large amounts of data at the genome, transcriptome, proteome and even metabolome level, and DNA and RNA cancer signature databases have been established. Here we present a database of differentially expressed proteins in human cancers (dbDEPC), with the goal of collecting curated cancer proteomics data, providing a resource for information on protein-level expression changes, and exploring protein profile differences among different cancers. dbDEPC currently contains 1803 proteins differentially expressed in 15 cancers, curated from 65 mass spectrometry (MS) experiments in peer-reviewed publications. In addition to MS experiments, low-throughput experiment data from the same literatures and cancer-associated genes from external databases were also integrated to provide some validation information. Furthermore, dbDEPC associates differential proteins with important structural variations in the human genome, such as copy number variations or single nucleotide polymorphisms, which might be helpful for explaining changes in protein expression at the DNA level. Data in dbDEPC can be queried by protein identifier, description or sequence; the retrieved protein entry provides the differential expression pattern seen in cancers, along with detailed annotations. dbDEPC is expected to be a reference database for cancer signatures at the protein level. This database is provided at http://dbdepc.biosino.org/index/.
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Affiliation(s)
- Hong Li
- Key Lab of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China
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Planque C, Kulasingam V, Smith CR, Reckamp K, Goodglick L, Diamandis EP. Identification of five candidate lung cancer biomarkers by proteomics analysis of conditioned media of four lung cancer cell lines. Mol Cell Proteomics 2009; 8:2746-58. [PMID: 19776420 DOI: 10.1074/mcp.m900134-mcp200] [Citation(s) in RCA: 114] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Detection of lung cancer at an early stage is necessary for successful therapy and improved survival rates. We performed a bottom-up proteomics analysis using a two-dimensional LC-MS/MS strategy on the conditioned media of four lung cancer cell lines of different histological backgrounds (non-small cell lung cancer: H23 (adenocarcinoma), H520 (squamous cell carcinoma), and H460 (large cell carcinoma); small cell lung cancer: H1688) to identify secreted or membrane-bound proteins that could be useful as novel lung cancer biomarkers. Proteomics analysis of the four conditioned media allowed identification of 1,830 different proteins (965, 871, 726, and 847 from H1688, H23, H460, and H520, respectively). All proteins were assigned a subcellular localization, and 38% were classified as extracellular or membrane-bound. We successfully identified the internal control proteins (also detected by ELISA), kallikrein-related peptidases 14 and 11, and IGFBP2. We also identified known or putative lung cancer tumor markers such as squamous cell carcinoma antigen, carcinoembryonic antigen, chromogranin A, creatine kinase BB, progastrin-releasing peptide, neural cell adhesion molecule, and tumor M2-PK. To select the most promising candidates for validation, we performed tissue specificity assays, functional classifications, literature searches for association to cancer, and a comparison of our proteome with the proteome of lung-related diseases and serum. Five novel lung cancer candidates, ADAM-17, osteoprotegerin, pentraxin 3, follistatin, and tumor necrosis factor receptor superfamily member 1A were preliminarily validated in the serum of patients with lung cancer and healthy controls. Our results demonstrate the utility of this cell culture proteomics approach to identify secreted and shed proteins that are potentially useful as serological markers for lung cancer.
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Affiliation(s)
- Chris Planque
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G1X5, Canada
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Yu KH, Barry CG, Austin D, Busch CM, Sangar V, Rustgi AK, Blair IA. Stable isotope dilution multidimensional liquid chromatography-tandem mass spectrometry for pancreatic cancer serum biomarker discovery. J Proteome Res 2009; 8:1565-76. [PMID: 19199705 PMCID: PMC2652408 DOI: 10.1021/pr800904z] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
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A novel approach to pancreatic cancer biomarker discovery has been developed, which employs a stable isotope labeled proteome (SILAP) standard coupled with extensive multidimensional separation coupled with tandem mass spectrometry (MS/MS). Secreted proteins from CAPAN-2 human pancreatic cancer derived cells were collected after conducting stable isotope labeling by amino acids in cell culture (SILAC). The resulting SILAP standard contained <0.5% of individual unlabeled proteins. Pooled sera from patients with early stage pancreatic cancer or controls were prepared, and an equal amount of the SILAP standard was added to each sample. Proteins were separated by isoelectric focusing (IEF) prior to two-dimensional liquid chromatography (2D-LC)–MS/MS analysis. A total of 1065 proteins were identified of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer. ELISA validation of these findings was successfully performed for two proteins, ICAM-1 and BCAM. Results of these studies have provided proof of principle that a SILAP standard derived from the CAPAN-2 secreted proteome can be used in combination with extensive multidimensional LC-MS/MS for the identification and relative quantitation of potential biomarkers of pancreatic cancer. This technique allows for the detection of low-abundance proteins, and focuses only on biologically relevant proteins derived from pancreatic cancer cells. CAPAN-2 human pancreatic ductal adenocarcinoma-derived cells were grown in [13C6, 15N1]leucine, [13C6,15N2]lysine. One-hundred twenty-one proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer. Results of these studies have provided proof of principle that a SILAP standard derived from the CAPAN-2 secreted proteome can be used in combination with extensive multidimensional LC−MS/MS for the identification and relative quantification of potential biomarkers of pancreatic cancer.
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Affiliation(s)
- Kenneth H Yu
- Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
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Abstract
Treatment of hematologic malignancies is evolving from a uniform approach to targeted therapies directed at the underlying molecular abnormalities of disease. The mixed lineage leukemia (MLL) proto-oncogene is a recurrent site of genetic rearrangements in acute leukemias; and since its discovery in 1992, many advances have been made in understanding its role in leukemogenesis. A variety of MLL translocation partners have been described, and detailed structure/function studies have identified functional domains that are required for transformation. Proteins associated with the MLL core complex or its fusion partners have been isolated and characterized for their critical roles in leukemia pathogenesis. Downstream mediators of MLL transcriptional regulation and multiple collaborating signaling pathways have been described and characterized. These advances in our understanding of MLL-related leukemogenesis provide a foundation for ongoing and future efforts to develop novel therapeutic strategies that will hopefully result in better treatment outcomes.
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Starita-Geribaldi M. Selection of pH ranges in 2DE. Methods Mol Biol 2009; 519:31-45. [PMID: 19381575 DOI: 10.1007/978-1-59745-281-6_3] [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: 05/27/2023]
Abstract
This chapter describes the technical improvements of the two-dimensional electrophoresis pattern resulting of an optimized pH range in the first dimension. Various types of pH gradients are available. Different strategies can be applied in order to select the pH ranges for the exploration of a proteome. The resulting gels are analysed for their background, resolution, sensitivity in relation with the sample complexity. As the complete dynamic range of protein expression cannot be visualized, the high loading capacity of immobilized narrow pH gradients can be used. The limitations and possible enhancements are discussed.
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Affiliation(s)
- Mireille Starita-Geribaldi
- Departement des Sciences Biologiques, UFR d'Odontologie, Pôle Universitaire Saint-Jean d'Angely, 24 avenue des Diables Bleus, 06357, Nice cedex, 4, France
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Abstract
Early diagnosis and prevention is a key factor in reducing the mortality and morbidity of cancer. However, currently available screening tools lack enough sensitivity for early diagnosis. It is important to develop noninvasive techniques and methods that can screen and identify asymptomatic patients who have cancer. Biomarkers of cancer status can also serve as powerful tools in monitoring the course of cancer and in determining the efficacy and safety of novel therapies. Thus, discovery of novel specific biomarkers are needed that may provide informative clues for early diagnosis and treatment of cancer. Recently, remarkable progress has been made in the development of new proteomics technology. The progress that has been made in this field is helpful in identifying biomarkers that can be used for early diagnosis of cancer and improving the understanding of the molecular etiological mechanism of cancer. This article describes the current state of the art in this field.
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Affiliation(s)
- Gary Guishan Xiao
- Osteoporosis Research Center, Departments of Medicine and Biomedical Sciences, Creighton University, 601 N 30 ST, Suite 6730, Omaha, NE 68131
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Chen EI, Yates JR. Cancer proteomics by quantitative shotgun proteomics. Mol Oncol 2007; 1:144-59. [PMID: 18443658 PMCID: PMC2352161 DOI: 10.1016/j.molonc.2007.05.001] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2007] [Revised: 04/30/2007] [Accepted: 05/01/2007] [Indexed: 11/30/2022] Open
Abstract
A major scientific challenge at the present time for cancer research is the determination of the underlying biological basis for cancer development. It is further complicated by the heterogeneity of cancer's origin. Understanding the molecular basis of cancer requires studying the dynamic and spatial interactions among proteins in cells, signaling events among cancer cells, and interactions between the cancer cells and the tumor microenvironment. Recently, it has been proposed that large-scale protein expression analysis of cancer cell proteomes promises to be valuable for investigating mechanisms of cancer transformation. Advances in mass spectrometry technologies and bioinformatics tools provide a tremendous opportunity to qualitatively and quantitatively interrogate dynamic protein-protein interactions and differential regulation of cellular signaling pathways associated with tumor development. In this review, progress in shotgun proteomics technologies for examining the molecular basis of cancer development will be presented and discussed.
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Affiliation(s)
- Emily I. Chen
- Department of Cell Biology, 10550 North Torrey Pines Road, SR11, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - John R. Yates
- Department of Cell Biology, 10550 North Torrey Pines Road, SR11, The Scripps Research Institute, La Jolla, CA 92037, USA
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Affiliation(s)
- Stephen W Hunsucker
- Department of Pediatrics, School of Medicine, University of Colorado at Denver and Health Sciences Center, 12801 East 17th Avenue, Aurora, CO 80010, USA
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