Selective enrichment and fractionation of phosphopeptides from peptide mixtures by isoelectric focusing after methyl esterification
Selective enrichment and fractionation of phosphopeptides from peptide mixtures by isoelectric focusing after methyl esterification
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DOI：
10.1021/ac061606u
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发表时间：
2007-03-01
影响因子：
7.4
通讯作者：
Neubert, Thomas A.
中科院分区：
文献类型：
--
作者：
Xu, Chong-Feng;Wang, Huaibin;Neubert, Thomas A.
我们开发了一种新的策略，根据甲酯化后等电点(PiS)的不同，从多肽混合物中富集和分级磷酸肽。α-S-酪蛋白和β-酪蛋白的混合物经甲基化胰酶等电聚焦后，磷酸小肽在酸性和中性PHS中被选择性地富集，而非磷酸小肽由于等电点高于固定化pH梯度的上限而离开聚焦胶。我们编写了一个基于网络的程序，pIM甲基化，用来预测有和没有甲酯化的多肽的等电点。PIM甲基化的理论计算表明，甲基化的磷酸肽和非磷酸肽可以根据每个肽中的磷酸基团和碱性残基的数量进行分组。我们的IEF结果与pIM甲基化计算的甲基化多肽的理论等电点基本一致。在正负离子模式下，2，6-二羟基苯乙酮均优于2，5-二羟基苯甲酸作为MALDI-Q-TOF MS的基质。
We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of alpha-S-casein and beta-casein, phosphopeptides were selectively enriched at acidic and neutral pHs while nonphosphopeptides left the focusing gel because their pIs are higher than the upper limit of the immobilized pH gradient. We wrote a web-based program, pIMethylation, to predict the pIs for peptides with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated phosphopeptides and non-phosphopeptides can be grouped on the basis of the number of phosphate groups and basic residues in each peptide. Our IEF results were consistent with theoretical pIs of methylated peptides calculated by pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes.