Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry

Jinn Shiun Chen, Kuei Tien Chen, Chung Wei Fan, Chia Li Han, Yu Ju Chen, Jau Song Yu, Yu Sun Chang, Chih Wei Chien, Chien Peng Wu, Ray Ping Hung, Err Cheng Chan

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)3028-3038
Number of pages11
JournalFEBS Journal
Volume277
Issue number14
DOIs
Publication statusPublished - Jul 2010
Externally publishedYes

Keywords

  • biomarker
  • colorectal cancer
  • mass spectrometry
  • membrane protein
  • proteomic profile

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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