Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and nanoLC-MS/MS analysis
Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and nanoLC-MS/MS analysis
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DOI：
10.1021/ac050232m
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发表时间：
2005-08-15
影响因子：
7.4
通讯作者：
Kramer, A
中科院分区：
文献类型：
--
作者：
Schlosser, A;Vanselow, JT;Kramer, A
我们开发了一种多蛋白酶方法，可以灵敏且全面地绘制蛋白质磷酸化位点。除胰蛋白酶外，低特异性蛋白酶弹性蛋白酶、蛋白酶 E 和嗜热菌蛋白酶的组合应用可产生高序列覆盖率，这是全面磷酸化位点定位的先决条件。使用最近推出的磷酸肽亲和材料钛球进行磷酸肽富集。我们优化了钛球富集磷酸肽的选择性，同时不影响接近 90% 的高回收率。使用高度灵敏的 nanoLC-MS/MS 系统使用 25-mu m-i.d. 分析富含磷酸肽的组分。反相柱，以 25 nL/min 的流速运行。新方法应用于小鼠昼夜节律蛋白周期 2 (mPER2)。通过多蛋白酶方法总共检测到了 mPER2 的 21 个磷酸化位点，而仅使用胰蛋白酶仅鉴定了 6 个磷酸化位点。事实证明，Titansphere 非常适合富集多种磷酸肽，包括携带两个、三个或四个磷酸化残基的肽，以及含有比酸性氨基酸更多的碱性氨基酸的磷酸肽。
We have developed a multi-protease approach that allows sensitive and comprehensive mapping of protein phosphorylation sites. The combined application of the low-specificity proteases elastase, proteinase E, and thermolysin in addition to trypsin results in high sequence coverage, a prerequisite for comprehensive phosphorylation site mapping. Phosphopeptide enrichment is performed with the recently introduced phosphopeptide affinity material titansphere. We have optimized the selectivity of the phosphopeptide enrichment with titansphere, without compromising the high recovery rate of similar to 90%. Phosphopeptide-enriched fractions are analyzed with a highly sensitive nanoLC-MS/MS system using a 25-mu m-i.d. reversed-phase column, operated at a flow rate of 25 nL/min. The new approach was applied to the murine circadian protein period 2 (mPER2). A total of 21 phosphorylation sites of mPER2 have been detected by the multi-protease approach, whereas only 6 phosphorylation sites were identified using solely trypsin. Titansphere proved to be well suited for the enrichment of a large variety of phosphopeptides, including peptides carrying two, three, or four phosphorylated residues, as well as phosphopeptides containing more basic than acidic amino acids.