title: An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer.
abstract: 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.
authors.affiliation: Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Chia-Li
authors.initials: CL
authors.lastname: Han
authors.forename: Jinn-Shiun
authors.initials: JS
authors.lastname: Chen
authors.forename: Err-Cheng
authors.initials: EC
authors.lastname: Chan
authors.forename: Chien-Peng
authors.initials: CP
authors.lastname: Wu
authors.forename: Kun-Hsing
authors.initials: KH
authors.lastname: Yu
authors.forename: Kuei-Tien
authors.initials: KT
authors.lastname: Chen
authors.forename: Chih-Chiang
authors.initials: CC
authors.lastname: Tsou
authors.forename: Chia-Feng
authors.initials: CF
authors.lastname: Tsai
authors.forename: Chih-Wei
authors.initials: CW
authors.lastname: Chien
authors.forename: Yung-Bin
authors.initials: YB
authors.lastname: Kuo
authors.forename: Pei-Yi
authors.initials: PY
authors.lastname: Lin
authors.forename: Jau-Song
authors.initials: JS
authors.lastname: Yu
authors.forename: Chuen
authors.initials: C
authors.lastname: Hsueh
authors.forename: Min-Chi
authors.initials: MC
authors.lastname: Chen
authors.forename: Chung-Chuan
authors.initials: CC
authors.lastname: Chan
authors.forename: Yu-Sun
authors.initials: YS
authors.lastname: Chang
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Chen
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D017314:Annexin A4 / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000378:metabolism*; D001798:Blood Proteins / Q000096:biosynthesis; D002272:Carcinoembryonic Antigen / Q000097:blood; D062465:Claudin-3; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D053208:Kaplan-Meier Estimate; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D015999:Multivariate Analysis; D010455:Peptides / Q000737:chemistry; D011379:Prognosis; D016016:Proportional Hazards Models; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D012372:ROC Curve; D053719:Tandem Mass Spectrometry / Q000379:methods; D023084:alpha-Defensins / Q000378:metabolism
chemical_list: D017314:Annexin A4; D014408:Biomarkers, Tumor; D001798:Blood Proteins; C568122:CLDN3 protein, human; D002272:Carcinoembryonic Antigen; D062465:Claudin-3; D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome; C406287:STOML2 protein, human; D023084:alpha-Defensins; C406413:human neutrophil peptide 1