Evaluation of the Low-Specificity Protease Elastase for Large-Scale Phosphoproteome Analysis
Evaluation of the Low-Specificity Protease Elastase for Large-Scale Phosphoproteome Analysis
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DOI：
10.1021/ac801708p
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发表时间：
2008-12-15
影响因子：
7.4
通讯作者：
Koerner, Roman
中科院分区：
文献类型：
--
作者：
Wang, Bin;Malik, Rainer;Koerner, Roman
全面的磷酸化位点定位是磷酸蛋白质组研究的中心目标，但使用单一的蛋白酶很少获得完整的蛋白质序列。在这项研究中，我们评估了与胰酶相比，使用弹性酶来增加从培养的人类细胞中浓缩的有丝分裂纺锤体蛋白的磷酸化位点覆盖率。我们利用Orbitrap质谱仪的高质量精度，通过分析弹性酶切割偏好和采用专门的两步数据库搜索策略，优化了数据库搜索的特异性。通过这种方法，我们从弹性酶消化的样品中检测到的磷酸化位点的数量大约翻了一番。值得注意的是，胰酶和弹性酶检测到的磷酸化位点具有很强的互补性，重叠不到10%。总体而言，我们使用胰酶确定了1068个磷酸化位点，使用弹性酶确定了467个磷酸化位点。经弹性酶消化后，约有30%的磷酸化位点被完全确定，证明了该酶在磷酸蛋白质组研究中的价值。
Comprehensive phosphorylation site mapping is the central goal of phosphoproteome studies, but complete protein sequence coverage is rarely obtained using one single protease. In this study, we have evaluated the use of elastase, in comparison to trypsin, to increase phosphorylation site coverage of mitotic spindle proteins enriched from cultured human cells. We took advantage of the high mass accuracy of Orbitrap mass spectrometers and optimized the database search specificity by analyzing both elastase cleavage preferences and employing a dedicated two-step database search strategy. Through this approach, we have approximately doubled the number of detectable phosphorylation sites from elastase digested samples. Remarkably, phosphorylation sites detected by trypsin and elastase were highly complementary with an overlap of less than 10%. In total, we identified 1068 phosphorylation sites using trypsin and 467 phosphorylation sites using elastase. Approximately 30% of the phosphorylation sites were exclusively identified after digestion by elastase, demonstrating the value of this enzyme for phosphoproteome studies.