Enhanced Detection of Multiply Phosphorylated Peptides and Identification of Their Sites of Modification

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• 作者：Antoine Fleitz ; Edward Nieves ; Carlos Madrid-Aliste ; Sarah J. Fentress ; L. David Sibley ; Louis M. Weiss ; Ruth Hogue Angeletti ; Fa-Yun Che
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：September 17, 2013
• 年：2013
• 卷：85
• 期：18
• 页码：8566-8576
• 全文大小：581K
• 年卷期：v.85,no.18(September 17, 2013)
• ISSN：1520-6882

文摘

Phosphorylation is an important post-translational modification that rapidly mediates many cellular events. A key to understanding the dynamics of the phosphoproteome is localization of the modification site(s), primarily determined using LC-MS/MS. A major technical challenge to analysis is the formation of phosphopeptide鈥搈etal ion complexes during LC which hampers phosphopeptide detection. We have devised a strategy that enhances analysis of phosphopeptides, especially multiply phosphorylated peptides. It involves treatment of the LC system with EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS, even if the column was treated with EDTA-Nab>2b> or if 25 mM EDTA-Nab>2b> was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of 伪-casein and 脽-casein were analyzed by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC to compare their performance in phosphopeptide analysis. With the first two approaches, no tri- and tetraphosphopeptides were identified in either 伪- or 脽-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the 伪-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the 脽-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 渭g of a human foreskin fibroblast cell lysate a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins were identified, and 2,164 unique phosphorylation sites were confidently localized (Ascore 鈮?0). Of these sites 79% were mono-, 20% di-, and 1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.