Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with Trypsin
Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with Trypsin
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DOI：
10.1021/acs.analchem.0c00478
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发表时间：
2020-07-21
影响因子：
7.4
通讯作者：
Rappsilber, Juri
中科院分区：
文献类型：
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作者：
Dau, Therese;Bartolomucci, Giulia;Rappsilber, Juri
胰蛋白酶是蛋白质组学中最常用的酶。然而，具有互补切割特异性的蛋白酶已被应用于特殊情况。在这项工作中，我们分析了五种蛋白酶替代胰蛋白酶的特性，用于蛋白质鉴定和序列覆盖时，应用于S。粟酒酵母全细胞裂解物。蛋白酶的特异性严重影响了鉴定的蛋白质的数量。具有较高特异性的蛋白酶比具有较低特异性的蛋白酶鉴定更多的蛋白质。然而，AspN、GluC、胰凝乳蛋白酶和蛋白酶K在很大程度上受益于在顺序消化中与胰蛋白酶配对，如我们之前对弹性蛋白酶所示。在最极端的情况下，用胰蛋白酶进行简化可使蛋白酶K鉴定的蛋白质数量提高731%。胰蛋白酶预消化也改善了其他蛋白酶的蛋白质鉴定，AspN（+62%），GluC（+80%）和胰凝乳蛋白酶（+21%）。有趣的是，用胰蛋白酶和AspN的顺序消化比单独用胰蛋白酶消化产生甚至更高数量的蛋白质鉴定。
Trypsin is the most used enzyme in proteomics. Nevertheless, proteases with complementary cleavage specificity have been applied in special circumstances. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted the number of proteins identified. Proteases with higher specificity led to the identification of more proteins than proteases with lower specificity. However, AspN, GIuC, chymotrypsin, and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, predigesting with trypsin improves the number of identified proteins for proteinase K by 731%. Trypsin predigestion also improved the protein identifications of other proteases, AspN (+62%), GIuC (+80%), and chymotrypsin (+21%). Interestingly, the sequential digest with trypsin and AspN yielded even a higher number of protein identifications than digesting with trypsin alone.