Fast-response proteomics by accelerated in-gel digestion of proteins
Fast-response proteomics by accelerated in-gel digestion of proteins
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DOI：
10.1021/ac026136s
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发表时间：
2003-03-15
影响因子：
7.4
通讯作者：
Shevchenko, A
中科院分区：
文献类型：
--
作者：
Havlis, J;Thomas, H;Shevchenko, A
以O-18标记的多肽为内标，采用MALDI TOF质谱法研究了改性胰蛋白酶和天然胰蛋白酶对蛋白质的胶内消化动力学。考察了酶解温度、酶浓度、酶解时间和凝胶块表面积对酶解产物产率的影响。基于动力学数据，我们开发了一种方案，该方案能够在30分钟消化时间内鉴定凝胶分离的蛋白质，而不影响肽产率和灵敏度，与通常依赖于过夜酶促裂解的常规方案相比。加速消化方案进行了测试，在120多个蛋白质的芽殖和裂殖酵母在亚皮摩尔水平的鉴定。
Kinetics of in-gel digestion of proteins by modified and native trypsins was studied by MALDI TOF mass spectrometry using O-18-labeled peptides as internal standards. The effect of the temperature, enzyme concentration, digestion time, and surface area of gel pieces on the yield of digestion products was characterized. Based on the kinetic data, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage. The accelerated digestion protocol was tested in identification of more than 120 proteins from budding and fission yeasts at the subpicomole level.