Enhanced Detection of Multiply Phosphorylated Peptides and Identification of Their Sites of Modification
Enhanced Detection of Multiply Phosphorylated Peptides and Identification of Their Sites of Modification
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DOI：
10.1021/ac401691g
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发表时间：
2013-09-17
影响因子：
7.4
通讯作者：
Che, Fa-Yun
中科院分区：
文献类型：
--
作者：
Fleitz, Antoine;Nieves, Edward;Che, Fa-Yun
磷酸化是一种重要的翻译后修饰，可迅速介导许多细胞事件。理解磷酸蛋白质组动力学的关键是修饰位点(S)的定位，这主要是通过LC-MS/MS来确定的。分析的主要技术挑战是在LC过程中形成磷肽-金属离子络合物，这阻碍了磷肽的检测。我们设计了一种策略来增强对磷酸肽的分析，特别是多个磷酸化肽。它包括用EDTA处理LC体系和2D-RP/RP-NanUPLC-MS/MS(高pH/低pH)分析。用EDTA-2D-RP/RP-NanUPLC-MS/MS在小于100fmolEDTA-2D-RP/RP-NanUPLC-MS/MS中检测到一种标准的三磷酸化肽，即使柱上用EDTA-Na-2处理或在样品中加入25 mM EDTA-Na-2，也不能检测到标准的三磷酸化肽。用前两种方法，在α-或β-酪蛋白样品中都没有鉴定出三磷和四磷。用EDTA-2D-RP/RP法在α-酪蛋白样品中鉴定出13个单磷酸、6个二磷酸和3个三磷酸多肽，而在β-酪蛋白样品中鉴定出19个单、8个二、4个三磷酸和3个四磷多肽。用EDTA-2D-RP/RP-NanUPLC-MS/MS检测500 mU g人包皮成纤维细胞裂解液，从1,087个独特的磷酸蛋白中鉴定出1,944个独特的磷酸肽，并确定了2,164个独特的磷酸化位点(ascore>；=20)。其中79%为单磷酸化，20%为双磷酸化，约1%为三磷酸化和四磷酸化，共鉴定出78个新的磷酸化位点。
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-metal 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-Na-2 or if 25 mM EDTA-Na-2 was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of alpha-casein and beta-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 alpha- or beta-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the alpha-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the beta-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 mu 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 >= 20). Of these sites 79% were mono-, 20% di-, and similar to 1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.