Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
Anal Chem. 2010 Dec 15;82(24):10095-101. doi: 10.1021/ac1020722. Epub 2010 Nov 15.
Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS(2) strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein.
尽管使用胰蛋白酶消化物的自下而上蛋白质组学被广泛用于定位蛋白质中的翻译后修饰（PTM），但在某些情况下，蛋白质在胰蛋白酶片段内有几个潜在的修饰位点，而 MS(2) 策略无法精确定位。我们在这里报告了一种使用两种蛋白酶（胰蛋白酶和胃蛋白酶）组合的方法，然后进行串联质谱分析，提供可以定位修饰位点的片段。我们使用该策略在玉米中表达的牛胰蛋白酶（TrypZean）上找到了一个糖基化位点。存在几种聚糖，并且全部都连接到蛋白质上的非共识 N-糖基化位点。
