Protein isoform-specific validation defines multiple chloride intracellular channel and tropomyosin isoforms as serological biomarkers of ovarian cancer
ABSTRACT New serological biomarkers for early detection and clinical management of ovarian cancer are urgently needed, and many candidates have been reported. A major challenge frequently encountered when validating candidates in patients is establishing quantitative assays that distinguish between highly homologous proteins. The current study tested whether multiple members of two recently discovered ovarian cancer biomarker protein families, chloride intracellular channel (CLIC) proteins and tropomyosins (TPM), were detectable in ovarian cancer patient sera. A multiplexed, label-free multiple reaction monitoring (MRM) assay was established to target peptides specific to all detected CLIC and TPM family members, and their serum levels were quantitated for ovarian cancer patients and non-cancer controls. In addition to CLIC1 and TPM1, which were the proteins initially discovered in a xenograft mouse model, CLIC4, TPM2, TPM3, and TPM4 were present in ovarian cancer patient sera at significantly elevated levels compared with controls. Some of the additional biomarkers identified in this homolog-centric verification and validation approach may be superior to the previously identified biomarkers at discriminating between ovarian cancer and non-cancer patients. This demonstrates the importance of considering all potential protein homologs and using quantitative assays for cancer biomarker validation with well-defined isoform specificity.
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ABSTRACT: Understanding the protection mechanism of 5'-AMP requires comprehensive knowledge of the proteins expressed during the period that the body is exposed to irradiation. Proteomics provides the tools for such analyses. Here, the experimental ICR mice were divided into three groups (normal group, model group and 5'-AMP + irradiation group). After different treatment, the hepatic total protein of each animal in three groups was separated by two-dimensional gel electrophoresis (2-DE). 2-DE analysis revealed fifty-eight protein spots were differentially expressed in comparison to three groups. From 58 protein spots, we selected nine spots to identify by MALDI-TOF-MS and received credible results. They were determined to be type I arginase, annexin A5, regucalcin, catalase, Tpm3 protein, Pdia4 protein, 14-3-3 protein epsilon, NAD-Malate dehydrogenase and heat shock protein 90. Considering the characteristic of these proteins, we proposed a possible protection pathway.International Journal of Molecular Sciences 12/2013; 15(1):186-202. DOI:10.3390/ijms15010186 · 2.86 Impact Factor
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ABSTRACT: Chemical crosslinking coupled with mass spectrometry provides structural information that is useful for probing protein conformations and providing experimental support for molecular models. "Zero-length" crosslinks have greater value for these applications than longer crosslinks because they provide more stringent distance constraints. However, this method is less commonly utilized because it cannot take advantage of isotopic labels, MS-labile bonds, or enrichment tags to facilitate identification. In this study, we combined label-free precursor ion quantitation and targeted tandem mass spectrometry with a new software tool, Zero-length Crosslink Miner (ZXMiner), to form a multi-tiered analysis strategy. A major, critical objective was to simultaneously achieve very high accuracy with essentially no false positive crosslink identifications, while maintaining a good depth of analysis. Our strategy was optimized on several proteins with known crystal structures. Comparison of ZXMiner to several existing crosslink analysis software showed that other algorithms detected less true positive crosslinks and were far less accurate. Although prior use of zero-length crosslinking was typically restricted to small proteins, ZXMiner and the associated strategy enables facile analysis of very large protein complexes. This was demonstrated by identification of zero-length crosslinks using purified 526 kDa spectrin heterodimers and intact red cell membranes and membrane skeletons.Journal of Proteome Research 12/2013; 13(2). DOI:10.1021/pr400953w · 5.00 Impact Factor
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ABSTRACT: A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories - structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins - 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 - were found to correlate with the corresponding proteins' abundance. The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins.Proteome Science 01/2014; 12(1):3. DOI:10.1186/1477-5956-12-3 · 1.88 Impact Factor