Quantitative Profiling of Drug-Associated Proteomic Alterations by Combined 2-Nitrobenzenesulfenyl Chloride (NBS) Isotope Labeling and 2DE/MS Identification

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• 作者：Keli Ou ; Djohan Kesuma ; Kumaresan Ganesan ; Kun Yu ; Sou Yen Soon ; Suet Ying Lee ; Xin Pei Goh ; Michelle Hooi ; Wei Chen ; Hiroyuki Jikuya ; Tetsuo Ichikawa ; Hiroki Kuyama ; Ei-ichi Matsuo ; Osamu Nishimura ; P
• 刊名：Journal of Proteome Research
• 出版年：2006
• 出版时间：September 2006
• 年：2006
• 卷：5
• 期：9
• 页码：2194 - 2206
• 全文大小：628K
• 年卷期：v.5,no.9(September 2006)
• ISSN：1535-3907

文摘

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal ofquantitative proteomics. Here, we describe a novel approach for identifying such drug-induced proteinalterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and controlsamples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and proteinalterations are identified by MS through the differential intensity of paired NBS peptide peaks. UsingNBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor(ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAMtreatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated withTAM or ER activity. We found the NBS labeling procedure to be both technically and biologicallyreproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DEdifferential protein quantitation, with discrepancies largely due to the comigration of distinct proteinsin the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78,CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4expression can serve as a novel prognostic factor for disease-free survival in two independent breastcancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes inbreast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator(SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellularprotein quantitation than conventional 2DE approaches.