Tài liệu Báo cáo khoa học: Comparison of membrane fraction proteomic profiles of normal and cancerous

Comparison of membrane fraction proteomic profiles of

normal and cancerous human colorectal tissues with

gel-assisted digestion and iTRAQ labeling mass

spectrometry

Jinn-Shiun Chen1,2, Kuei-Tien Chen3

, Chung-Wei Fan2,4, Chia-Li Han5

, Yu-Ju Chen5

, Jau-Song Yu6

,

Yu-Sun Chang7

, Chih-Wei Chien5

, Chien-Peng Wu5

, Ray-Ping Hung3 and Err-Cheng Chan3

1 Department of Surgery, Chang Gung Memorial Hospital, Tao Yuan, Taiwan

2 College of Medicine, Chang Gung University, Tao Yuan, Taiwan

3 Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Tao Yuan, Taiwan

4 Department of Colorectal Surgery, Chang Gung Memorial Hospital, Keelung, Taiwan

5 Institute of Chemistry, Academia Sinica, Taipei, Taiwan

6 Department of Cell and Molecular Biology, Chang Gung University, Tao Yuan, Taiwan

7 Molecular Medicine Research Center, Chang Gung University, Tao Yuan, Taiwan

Introduction

Colorectal cancer (CRC) remains one of the most

prevalent cancers in the western world and the third

highest cause of cancer mortality in Taiwan [1]. CRC

is thought to evolve into invasive cancer from adeno￾Keywords

biomarker; colorectal cancer; mass

spectrometry; membrane protein; proteomic

profile

Correspondence

E.-C. Chan, Department of Medical

Biotechnology and Laboratory Science,

Chang Gung University, 259 Wen-Hua 1st

Road, Kweishan, Taoyuan, Taiwan, China

Fax: +886 3 2118741

Tel: +886 3 2118800 (ext. 5220)

E-mail: [email protected]

Note

Jinn-Shiun Chen, Kuei-Tien Chen and

Chung-Wei Fan contributed equally to this

article

(Received 12 January 2010, revised 9 April

2010, accepted 17 May 2010)

doi:10.1111/j.1742-4658.2010.07712.x

The aim of this study was to uncover the membrane protein profile differ￾ences 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 fundamen￾tal 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 quantifi￾cation, 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 pro￾teins 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 mito￾chondrial solute carrier family 25A4 were confirmed by western blotting.

We conclude that gel-assisted digestion and iTRAQ labeling MS is a poten￾tial approach for uncovering and comparing membrane protein profiles of

tissue samples that has the potential to identify novel biomarkers.

Abbreviations

CEA, carcinoembryonic antigen-related cell adhesion molecule 5; CLDN, claudin; CLDN3, claudin-3; CLDN4, cluadin-4; CRC, colorectal

carcinoma; HLA, human leukocyte antigen; HLA-A1, HLA class I histocompatibility antigen A-1; iTRAQ, isobaric tags with related and

absolute quantitation; SLC25A4, mitochondrial solute carrier family 25A4; TAPBP, tapasin.

3028 FEBS Journal 277 (2010) 3028–3038 ª 2010 The Authors Journal compilation ª 2010 FEBS