Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis Applying Dual-Step 18O Labeling Coupled with Immobilized Metal-Ion Affinity Chromatography

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• 作者：Shi-Jian Ding ; Yingchun Wang ; Jon M. Jacobs ; Wei-Jun Qian ; Feng Yang ; Aleksey V. Tolmachev ; Xiuxia Du ; Wei Wang ; Ronald J. Moore ; Matthew E. Monroe ; Samuel O. Purvine ; Katrina Waters ; Tyler H. Heibeck
• 刊名：Journal of Proteome Research
• 出版年：2008
• 出版时间：October 3, 2008
• 年：2008
• 卷：7
• 期：10
• 页码：4215-4224
• 全文大小：321K
• 年卷期：v.7,no.10(October 3, 2008)
• ISSN：1535-3907

文摘

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed ¹⁶O/¹⁸O labeling plus ¹⁶O/¹⁸O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC−MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.