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.
This manuscript addresses the importance of distinguishing between protein homologs and isoforms when identifying and validating cancer biomarkers in plasma or serum. Specifically, it describes the use of targeted in-depth LC-MS/MS analysis to determine the members of two protein families, chloride intracellular channel (CLIC) and tropomyosin (TPM) proteins that are detectable in sera of ovarian cancer patients. It then establishes a multiplexed isoform- and homology-specific MRM assay to quantify all observed gene products in these two protein families as well as many of the closely related tropomyosin isoforms. Using this assay, levels of all detected CLICs and TPMs were quantified in ovarian cancer patient and control subject sera. These results demonstrate that in addition to the previously known CLIC1, multiple tropomyosins and CLIC4 are promising new ovarian cancer biomarkers. Based on these initial validation studies, these new ovarian cancer biomarkers appear to be superior to most previously known ovarian cancer biomarkers.
"The list was filtered for known cancer biomarkers. CS and ES shared four known cancer biomarkers (hemoglobin alpha 1/2 [HBA1/2], galectin- 3-binding protein [LGACS3BP], serum amyloid A1 [SAA1], and thrombospondin [THBS1])     , and separately they had three (APOE, tropomyosin alpha-3 chain [TPM3], and transthyretin [TTR])    and one (C-reactive protein [CRP])  marker(s), respectively (Fig. 1C). APOE was selected for further verification because it has been shown to be upregulated in various cancers including lung cancer  . "
[Show abstract][Hide abstract] ABSTRACT: Biomarkers to identify subjects at high-risk for developing lung cancer will revolutionize the disease outlook. Most biomarker studies have focused on patients already diagnosed with lung cancer and in most cases the disease is often advanced and incurable. The objective of this study was to use proteomics to identify a plasma biomarker for early detection of lung lesions that may subsequently be the harbinger for cancer. Plasma samples were obtained from subjects without lung cancer grouped as never, current, or ex-smokers. An iTRAQ-based proteomic analysis was performed on these pooled plasma samples. We identified 31 proteins differentially abundant in current smokers or ex-smokers relative to never smokers. Western blot and ELISA analyses confirmed the iTRAQ results which demonstrated an increase of apolipoprotein E (APOE) in current smokers as compared to both never and ex-smokers. There was a strong and significant correlation of the plasma APOE levels with development of pre-malignant squamous metaplasia. Additionally, we also showed that higher tissue levels of APOE are seen with squamous metaplasia, supporting a direct relationship. Our analysis reveals that elevated plasma APOE is associated with smoking, and APOE is a novel predictive protein biomarker for early morphological changes of squamous metaplasia in the lung. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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 · 4.25 Impact Factor
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