Improved Sensitivity for Phosphopeptide Mapping Using Capillary Column HPLC and Microionspray Mass Spectrometry: Comparative Phosphorylation Site Mapping from Gel-Derived Proteins

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• 作者：Francesca Zappacosta ; Michael J. Huddleston ; Ryan L. Karcher ; Vladimir I. Gelfand ; Steven A. Carr ; and Roland S. Annan
• 刊名：Analytical Chemistry
• 出版年：2002
• 出版时间：July 1, 2002
• 年：2002
• 卷：74
• 期：13
• 页码：3221 - 3231
• 全文大小：195K
• 年卷期：v.74,no.13(July 1, 2002)
• ISSN：1520-6882

文摘

Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylationcontrols a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated ona given protein. This is often a nontrivial task. In additionto the difficulties involved in purifying sufficient amountsof any given protein, most phosphoproteins containmultiple, substoichiometric sites of phosphorylation. Inthis paper, we describe substantial improvements madeto our previously reported multidimensional electrosprayMS-based phosphopeptide mapping technique that haveresulted in a 20-fold increase in sensitivity for the overallprocess. Chief among these improvements are the incorporation of capillary chromatography and a microionspraysource for the mass spectrometer into the first dimensionof the analysis. In the first dimension of the process,phosphopeptides present in the proteolytic digest of aprotein are selectively detected and collected into fractionsduring on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ionscanning. The relative merits and limitations of these twotechniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the methodunder the new experimental conditions makes it suitablefor phosphorylation mapping (from selective detectionthrough sequencing) on gel-separated phosphoproteinswhere the level of phosphorylation at any given site is<200 fmol. Furthermore, this method detects serine,threonine, and tyrosine phosphorylation equally well. Wehave successfully employed this new configuration to map11 in vivo sites of phosphorylation on the Saccharomycescerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate forPKA. In addition, four peptides containing cdk sites andthe autophosphorylation site at Tyr⁵³⁰ were found to bephosphorylated. Because the first dimension of thismethod generates a phosphorylation profile that can beused for a semiquantitative evaluation of site specificphosphorylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a proteinin response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detecta change in phosphorylation stoichiometry on the motorprotein myosin-V in response to treatment with eithermitotic or interphase Xenopus egg extracts and to identifythe single functionally significant phosphorylation site thatregulates myosin-V cargo binding.