Open Tubular Immobilized Metal Ion Affinity Chromatography Combined with MALDI MS and MS/MS for Identification of Protein Phosphorylation Sites

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• 作者：Huaizhi Liu ; Jacek Stupak ; Jing Zheng ; Bernd O. Keller ; Brenda J. Brix ; Larry Fliegel ; and Liang Li
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：July 15, 2004
• 年：2004
• 卷：76
• 期：14
• 页码：4223 - 4232
• 全文大小：174K
• 年卷期：v.76,no.14(July 15, 2004)
• ISSN：1520-6882

文摘

Protein phosphorylation is one of the most importantknown posttranslational modifications. Tandem massspectrometry has become an important tool for mappingout the phosphorylation sites. However, when a peptidegenerated from the enzymatic or chemical digestion of aphosphoprotein is highly phosphorylated or containsmany potential phosphorylation residues, phosphorylationsite assignment becomes difficult. Separation and enrichment of phosphopeptides from a digest mixture is desirable and often a critical step for MS/MS-based sitedetermination. In this work, we present a novel opentubular immobilized metal ion affinity chromatography(OT-IMAC) method, which is found to be more effectiveand reproducible for phosphopeptide enrichment, compared to a commonly used commercial product, Ziptipfrom Millipore. A strategy based on a combination of OT-IMAC, sequential dual-enzyme digestion, and matrix-assisted laser desorption/ionization (MALDI) quadrupoletime-of-flight tandem mass spectrometry for phosphoprotein characterization is presented. It is shown thatMALDI MS/MS with collision-induced dissociation canbe very effective in generating fragment ion spectracontaining rich structural information, which enables theidentification of phosphorylation sites even from highlyphosphorylated peptides. The applicability of this methodfor real world applications is demonstrated in the characterization and identification of phosphorylation sites ofa Na⁺/H⁺ exchanger fusion protein, His182, which wasphosphorylated in vitro using the kinase Erk2.