An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer

• Published in: Molecular & cellular proteomics 2011-04, Vol.10 (4), p.M110.003087-M110.003087, Article M110.003087
• Main Authors: Han, Chia-Li, Chen, Jinn-Shiun, Chan, Err-Cheng, Wu, Chien-Peng, Yu, Kun-Hsing, Chen, Kuei-Tien, Tsou, Chih-Chiang, Tsai, Chia-Feng, Chien, Chih-Wei, Kuo, Yung-Bin, Lin, Pei-Yi, Yu, Jau-Song, Hsueh, Chuen, Chen, Min-Chi, Chan, Chung-Chuan, Chang, Yu-Sun, Chen, Yu-Ju
• Format: Article
• Language: English
• Subjects: Adenocarcinoma - diagnosis; Adenocarcinoma - metabolism; Adenocarcinoma - pathology; Adult; Aged; Aged, 80 and over; alpha-Defensins - metabolism; Amino Acid Sequence; Annexin A4 - metabolism; Biomarkers, Tumor - chemistry; Biomarkers, Tumor - metabolism; Blood Proteins - biosynthesis; Carcinoembryonic Antigen - blood; Claudin-3; Colorectal Neoplasms - diagnosis; Colorectal Neoplasms - metabolism; Colorectal Neoplasms - pathology; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Membrane Proteins - biosynthesis; Membrane Proteins - blood; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Middle Aged; Molecular Diagnostic Techniques; Multivariate Analysis; Peptides - chemistry; Prognosis; Proportional Hazards Models; Proteome - chemistry; Proteome - metabolism; ROC Curve; Tandem Mass Spectrometry - methods
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M110.003087

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48–70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.