Increasing phosphoproteome coverage and identification of phosphorylation motifs through combination of different HPLC fractionation methods
Increasing phosphoproteome coverage and identification of phosphorylation motifs through combination of different HPLC fractionation methods
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DOI：
10.1016/j.jchromb.2010.11.004
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发表时间：
2011-01-01
影响因子：
3
通讯作者：
Guo, Lin
中科院分区：
文献类型：
--
作者：
Chen, Xi;Wu, Di;Guo, Lin
蛋白质磷酸化激活或失活许多其他蛋白质，特别是蛋白酶，并在广泛的细胞过程中发挥重要作用。磷酸肽富集程序和基于质谱的肽测序技术的最新进展使我们能够识别大量的蛋白质磷酸化位点。在这项研究中，我们结合了三种不同的HPLC技术在分馏富集的磷酸肽RPLC-MS/MS分析，并发现，虽然4000-5000之间的独特的磷酸肽可以确定以下的任何HPLC馏分方法，不同的HPLC方法产生了相当数量的非重叠的独特的磷酸肽。结合所有HPLC方法的数据，我们能够识别9069个独特的磷酸肽和3260个磷酸蛋白，覆盖9463个独特的磷酸化位点，表明不同的HPLC方法是互补的，可以一起使用，以增加磷酸蛋白质组的覆盖率。我们的研究还发现了一些新的磷酸化位点和新的磷酸化模体。(C)2010爱思唯尔有限公司版权所有。
Protein phosphorylation activates or deactivates many other proteins especially protein enzymes, and plays a significant role in a wide range of cellular processes. Recent advances in phosphopeptide enrichment procedures and mass spectrometry-based peptide sequencing techniques have enabled us to identify large number of protein phosphorylation sites. In this study, we combined three different HPLC techniques in fractionating enriched phosphopeptides before RPLC-MS/MS analysis, and found that although between 4000-5000 unique phosphopeptides could be identified following any of the HPLC fraction method, different HPLC method yielded a considerable amount of non-overlapping unique phosphopeptides. Combining data from all the HPLC methods, we were able to identify 9069 unique phosphopeptides and 3260 phosphoproteins covering 9463 unique phosphorylation sites, indicating that different HPLC methods are complementary to each other, and can be used together in order to increase the phosphoproteome coverage. A number of new phosphorylation sites and novel phosphorylation motifs were also discovered from our study. (C) 2010 Elsevier B.V. All rights reserved.