Product(s) used in this publication: Reference Peptides for Targeted Proteomics - SpikeTides™ & SpikeMix™
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 (SRM/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 (upregulation of ANP32A, ANXA3, SORD, LDHA, LCN2, NCL, S100A11, SERPINB5, CDV3, OLFM4, and REG4; downregulation of ARF6 and PGM5), and a five-protein 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 upregulation; KCTD12 downregulation) or adenocarcinoma (ANP32B, G6PD, RCN1, and SET upregulation; 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 investment-worthy biomarker candidates that can be used to develop a standardized pre-colonoscopy blood test for the early detection of colorectal tumors.