Identification of Phosphorylation Sites in Proteins Separated by Polyacrylamide Gel Electrophoresis

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• 作者：Xiaolong Zhang ; Christopher J. Herring ; Patrick R. Romano ; Joanna Szczepanowska ; Hanna Brzeska ; Alan G. Hinnebusch ; and Jun Qin
• 刊名：Analytical Chemistry
• 出版年：1998
• 出版时间：May 15, 1998
• 年：1998
• 卷：70
• 期：10
• 页码：2050 - 2059
• 全文大小：171K
• 年卷期：v.70,no.10(May 15, 1998)
• ISSN：1520-6882

文摘

We report a fast, sensitive, and robust procedure for theidentification of precise phosphorylation sites inproteinsseparated by polyacrylamide gel electrophoresis by acombination of matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI/TOF) and on-line capillary liquid chromatography electrospray tandemion trap mass spectrometry (LC/ESI/MS/MS). With thisprocedure, a single phosphorylation site was identifiedon as little as 20 ng (500 fmol) of the baculovirus-expressed catalytic domain of myosin I heavy-chain kinaseseparated by gel electrophoresis. The phosphoproteinisdigested in the gel with trypsin, and the resultingpeptidesare extracted with >60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase toidentify the phosphopeptides. The phosphopeptides arethen separated and fragmented in an on-line LC/ESI iontrap mass spectrometer to identify the precise phosphorylation sites. This procedure eliminates any off-lineHPLC separation and minimizes sample handling. Theuse of MALDI/TOF and LCQ, two types of mass spectrometers that are widely available to the biological community, will make this procedure readily accessible tobiologists. We applied this technique to identify twoautophosphorylation sites and to assign at least another12 phosphorylation sites to two tryptic peptides in aseriesof experiments using a gel slice containing only 200 ng(3 pmol) of human double-stranded RNA-activated protein kinase expressed in a mutant strain of the yeastSaccharomyces cerevisiae.