Han, Chia-LiChia-LiHanChen, Jinn-ShiunJinn-ShiunChenChan, Err-ChengErr-ChengChanWu, Chien-PengChien-PengWuYu, Kun-HsingKun-HsingYuChen, Kuei-TienKuei-TienChenTsou, Chih-ChiangChih-ChiangTsouTsai, Chia-FengChia-FengTsaiChien, Chih-WeiChih-WeiChienKuo, Yung-BinYung-BinKuoLin, Pei-YiPei-YiLinYu, Jau-SongJau-SongYuHsueh, ChuenChuenHsuehChen, Min-ChiMin-ChiChenChan, Chung-ChuanChung-ChuanChanChang, Yu-SunYu-SunChangChen, Yu-JuYu-JuChen2012-05-122018-07-102012-05-122018-07-102011http://ntur.lib.ntu.edu.tw//handle/246246/239171We 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. ? 2011 by The American Society for Biochemistry and Molecular Biology, Inc.en-US[SDGs]SDG3alpha defensin; annexin; carcinoembryonic antigen; claudin 3; membrane protein; stomatin; stomatin like 2; tumor marker; unclassified drug; alpha defensin; carcinoembryonic antigen; claudin 3; defensin 1; lipocortin 4; membrane protein; peptide; plasma protein; proteome; STOML2 protein, human; tumor marker; accuracy; adult; article; bioinformatics; cancer diagnosis; cancer recurrence; cancer survival; colorectal cancer; controlled study; early diagnosis; enzyme linked immunosorbent assay; female; fractionation; high performance liquid chromatography; human; human tissue; major clinical study; male; mass spectrometry; priority journal; protein analysis; protein expression; proteomics; serology; adenocarcinoma; aged; amino acid sequence; biosynthesis; blood; chemistry; colorectal tumor; gene expression regulation; Kaplan Meier method; metabolism; methodology; middle aged; molecular diagnosis; multivariate analysis; pathology; prognosis; proportional hazards model; receiver operating characteristic; tandem mass spectrometry; Adenocarcinoma; Adult; Aged; Aged, 80 and over; alpha-Defensins; Amino Acid Sequence; Annexin A4; Blood Proteins; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Membrane Proteins; Middle Aged; Molecular Diagnostic Techniques; Multivariate Analysis; Peptides; Prognosis; Proportional Hazards Models; Proteome; ROC Curve; Tandem Mass Spectrometry; Tumor Markers, Biologicaljournal article10.1074/mcp.M110.003087http://ntur.lib.ntu.edu.tw/bitstream/246246/239171/-1/157.pdf