Gauci Sharon, Helbig Andreas O, Slijper Monique, Krijgsveld Jeroen, Heck Albert J R, Mohammed Shabaz
Biomolecular Mass Spectrometry and Proteomics Group, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Anal Chem. 2009 Jun 1;81(11):4493-501. doi: 10.1021/ac9004309.
The analysis of proteome-wide phosphorylation events is still a major analytical challenge because of the enormous complexity of protein phosphorylation networks. In this work, we evaluate the complementarity of Lys-N, Lys-C, and trypsin with regard to their ability to contribute to the global analysis of the phosphoproteome. A refined version of low-pH strong cation exchange was used to efficiently separate N-terminally acetylated, phosphorylated, and nonmodified peptides. A total of 5036 nonredundant phosphopeptides could be identified with a false discovery rate of <1% from 1 mg of protein using a combination of the three enzymes. Our data revealed that the overlap between the phosphopeptide data sets generated with different proteases was marginal, whereas the overlap between two similarly generated tryptic data sets was found to be at least 4 times higher. In this way, the parallel use of Lys-N and trypsin enabled a 72% increase in the number of detected phosphopeptides as compared to trypsin alone, whereas a trypsin replicate experiment only led to a 25% increase. Thus, when focusing solely on the trypsin and Lys-N data, we identified 4671 nonredundant phosphopeptides. Further analysis of the detected sites showed that the Lys-N and trypsin data sets were enriched in significantly different phosphorylation motifs, further evidencing that multiprotease approaches are very valuable in phosphoproteome analyses.
由于蛋白质磷酸化网络极其复杂，对全蛋白质组磷酸化事件进行分析仍是一项重大的分析挑战。在本研究中，我们评估了赖氨酰内切酶N（Lys-N）、赖氨酰内切酶C（Lys-C）和胰蛋白酶在助力磷酸化蛋白质组全局分析方面的互补性。使用低pH强阳离子交换的优化版本来有效分离N端乙酰化、磷酸化和未修饰的肽段。使用这三种酶组合，从1毫克蛋白质中可鉴定出总共5036个非冗余磷酸肽，错误发现率<1%。我们的数据显示，不同蛋白酶生成的磷酸肽数据集之间的重叠很少，而两个类似生成的胰蛋白酶数据集之间的重叠至少高出4倍。通过这种方式，与单独使用胰蛋白酶相比，并行使用Lys-N和胰蛋白酶可使检测到的磷酸肽数量增加72%，而胰蛋白酶重复实验仅导致增加25%。因此，仅关注胰蛋白酶和Lys-N数据时，我们鉴定出4671个非冗余磷酸肽。对检测到的位点进行进一步分析表明，Lys-N和胰蛋白酶数据集富含显著不同的磷酸化基序，进一步证明多蛋白酶方法在磷酸化蛋白质组分析中非常有价值。
