Targeted Proteomics for Multiplexed Verification of Markers of Colorectal Tumorigenesis

Abstract

Targeted proteomic methods can accelerate the verification of multiple tumor marker candidates in large series of patient samples. We utilized the targeted approach known as selected/multiple reaction monitoring (S/MRM) to verify potential protein markers of colorectal adenoma identified by our group in previous transcriptomic and quantitative shotgun proteomic studies of a large cohort of precancerous colorectal lesions. We developed SRM assays to reproducibly detect and quantify 25 (62.5%) of the 40 selected proteins in an independent series of precancerous and cancerous tissue samples (19 adenoma/ normal mucosa pairs; 17 adenocarcinoma/normal mucosa pairs). Twenty-three proteins were significantly upregulated (n ‫؍‬ 17) or downregulated (n ‫؍‬ 6) in adenomas and/or adenocarcinomas, as compared with normal mucosa (linear fold changes > ؎1.3, adjusted p value <0.05). Most changes were observed in both tumor types (up-regulation of ANP32A, ANXA3, SORD, LDHA, LCN2, NCL, S100A11, SERPINB5, CDV3, OLFM4, and REG4; downregulation of ARF6 and PGM5), and a fiveprotein biomarker signature distinguished neoplastic tissue from normal mucosa with a maximum area under the receiver operating curve greater than 0.83. Other changes were specific for adenomas (PPA1 and PPA2 up-regulation; KCTD12 downregulation) or adenocarcinoma (ANP32B, G6PD, RCN1, and SET up-regulation; downregulated AKR1B1, APEX1, and PPA1). Some changes significantly correlated with a few patient-or tumor-related phenotypes. Twenty-two (96%) of the 23 proteins have a potential to be released from the tumors into the bloodstream, and their detectability in plasma has been previously reported. The proteins identified in this study expand the pool of biomarker candidates that can be used to develop a standardized precolonoscopy blood test for the early detection of colorectal tumors. Molecular & Cellular

Olfactomedin 4 downregulation is associated with tumor initiation, growth and progression in human prostate cancer

“…20 The OLFM4 gene has been analyzed as a putative biomarker in many cancers, including gastrointestinal cancer, head and neck squamous cell carcinoma, cervical neoplasia, nonsmall cell lung cancer, triple-negative breast cancer and distant metastases in estrogen receptor-positive breast carcinoma. [20][21][22][23][24][25][26][27][28][29][30][31][32] In our study, we provide clinical evidence that reduced OLFM4 expression was associated with prostate cancer progression and with DNA methylation of CpG sites in the OLFM4 gene promoter region in human prostate adenocarcinoma. We also found that OLFM4 may play a role in regulating EMT, as well as tumor initiation and growth, in prostate cells.…”

“…In addition, DNA methylation of the OLFM4 gene has been found to be associated with tumor aggressiveness and patient outcomes in gastric carcinoma . The OLFM4 gene has been analyzed as a putative biomarker in many cancers, including gastrointestinal cancer, head and neck squamous cell carcinoma, cervical neoplasia, nonsmall cell lung cancer, triple‐negative breast cancer and distant metastases in estrogen receptor‐positive breast carcinoma …”

ExSTA: External Standard Addition Method for Accurate High‐Throughput Quantitation in Targeted Proteomics Experiments

“…Although relative quantitation techniques are still in use and are still valuable for biomarker discovery, for biomarker verification, and clinical analysis, what is required are techniques that allow determination of the absolute amount of material present, so that the amount of protein in a patient sample analyzed in various clinical laboratories can be compared with normal levels. Although these quantitation techniques are sometimes called “absolute”, the concentrations in the biological sample are, in fact, determined by comparison to known amounts of standard materials . Exactly how this comparison is performed is the subject of this current paper.…”

Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection of Colorectal Cancer

“…In addition to histological and plasma samples, S100A8 and S100A9 have been shown to be overexpressed in fecal samples also using a LC-MS/MS approach [118,119]. Additionally, S100A11 has been identified among a cohort of 23 upregulated proteins in CRC samples using a combined targeted LC-MS/MS and SRM approach [22].…”

“…Over the last 2 decades, unprecedented technological advancement in proteinbased mass spectrometry (proteomics) has radically changed the landscape of biomarker research [13] (Table 1). This has facilitated the characterization of complex cellular proteomes [14][15][16][17][18][19], research that has identified hundreds of over and under expressed proteins in carcinoma patients using tumor tissue, histological sections, plasma or fecal samples when compared to matched normal tissues [20][21][22][23][24]. Despite this, with the exception of Carcinoembryonic antigen (CEA) and Cancer antigen [25], no new protein biomarkers have made it into routine clinical practice [21,26,27].…”