Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry.
The aim of this study was to uncover the membrane protein profile differences between colorectal carcinoma and neighboring normal mucosa from colorectal cancer patients. Information from cellular membrane proteomes can be used not only to study the roles of membrane proteins in fundamental biological processes, but also to discover novel targets for improving the management of colorectal cancer patients. We used solvent extraction and a gel-assisted digestion method, together with isobaric tags with related and absolute quantitation (iTRAQ) reagents to label tumoral and adjacent normal tissues in a pairwise manner (n = 8). For high-throughput quantification, these digested labeled peptides were combined and simultaneously analyzed using LC-MS/MS. Using the shotgun approach, we identified a total of 438 distinct proteins from membrane fractions of all eight patients. After comparing protein expression between cancerous and corresponding normal tissue, we identified 34 upregulated and eight downregulated proteins with expression changes greater than twofold (Student's t-test, P < 0.05). Among these, the overexpression of well-established biomarkers such as carcinoembryonic antigens (CEACAM5, CEACAM6), as well as claudin-3, HLA class I histocompatibility antigen A-1, tapasin and mitochondrial solute carrier family 25A4 were confirmed by western blotting. We conclude that gel-assisted digestion and iTRAQ labeling MS is a potential approach for uncovering and comparing membrane protein profiles of tissue samples that has the potential to identify novel biomarkers.
Chen
Jinn-Shiun
JS
Department of Surgery, Chang Gung Memorial Hospital, Tao Yuan, Taiwan.
Chen
Kuei-Tien
KT
Fan
Chung-Wei
CW
Han
Chia-Li
CL
Chen
Yu-Ju
YJ
Yu
Jau-Song
JS
Chang
Yu-Sun
YS
Chien
Chih-Wei
CW
Wu
Chien-Peng
CP
Hung
Ray-Ping
RP
Chan
Err-Cheng
EC
D033741:Adenine Nucleotide Translocator 1 / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D062465:Claudin-3; D016000:Cluster Analysis; D003106:Colon / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism; D015788:HLA-A1 Antigen / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012007:Rectum / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
D033741:Adenine Nucleotide Translocator 1; D015415:Biomarkers; C568122:CLDN3 protein, human; D002272:Carcinoembryonic Antigen; D062465:Claudin-3; D015788:HLA-A1 Antigen; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; C107762:tapasin; D014357:Trypsin