Wang Bin, Malik Rainer, Nigg Erich A, Körner Roman
Department of Cell Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Anal Chem. 2008 Dec 15;80(24):9526-33. doi: 10.1021/ac801708p.
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.
全面的磷酸化位点图谱绘制是磷酸化蛋白质组研究的核心目标，但使用单一蛋白酶很少能实现完整的蛋白质序列覆盖。在本研究中，我们评估了与胰蛋白酶相比，使用弹性蛋白酶来增加从培养的人类细胞中富集的有丝分裂纺锤体蛋白的磷酸化位点覆盖范围。我们利用了轨道阱质谱仪的高质量精度，并通过分析弹性蛋白酶的切割偏好和采用专门的两步数据库搜索策略来优化数据库搜索特异性。通过这种方法，我们使弹性蛋白酶消化样品中可检测到的磷酸化位点数量增加了约一倍。值得注意的是，胰蛋白酶和弹性蛋白酶检测到的磷酸化位点具有高度互补性，重叠率不到10%。我们总共使用胰蛋白酶鉴定出1068个磷酸化位点，使用弹性蛋白酶鉴定出467个磷酸化位点。约30%的磷酸化位点是在弹性蛋白酶消化后才被专门鉴定出来的，这证明了这种酶在磷酸化蛋白质组研究中的价值。
