An improved trypsin digestion method minimizes digestion-induced modifications on proteins
An improved trypsin digestion method minimizes digestion-induced modifications on proteins
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DOI：
10.1016/j.ab.2009.05.018
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发表时间：
2009-09-01
影响因子：
2.9
通讯作者：
Bondarenko, Pavel V.
中科院分区：
文献类型：
--
作者：
Ren, Da;Pipes, Gary D.;Bondarenko, Pavel V.
胰蛋白酶消化可诱导人工修饰，例如由于消化过程中使用的温度和碱性pH缓冲液，蛋白质上的天冬酰胺脱酰胺和N-末端谷氨酰胺环化。这些人工修饰的量与蛋白质样品在还原/烷基化缓冲液中的孵育时间成正比，更重要的是，与肽完全暴露于溶剂的消化缓冲液中的孵育时间成正比。为了使这些人工修饰最小化，我们将重点放在通过使胰蛋白酶活性最大化来使胰蛋白酶消化时间最小化。通过从消化缓冲液中完全去除胍（一种已知的胰蛋白酶抑制剂）来优化胰蛋白酶活性。结果，在30 min内实现了油减少和烷基化免疫球蛋白γ分子的接近完全的胰蛋白酶消化。使用在线LTQ Orbitrap质谱仪通过反相液相色谱/串联质谱法分析和定量了蛋白质胰蛋白酶片段及其修饰产物。还通过使用高分辨率飞行时间质谱仪监测反应的完全性来最小化还原和烷基化反应时间。使用这种30分钟溶液中胰蛋白酶消化方法，观察到很少的方案诱导的脱酰胺或N-末端谷氨酰胺环化产物，并且由于较少的胰蛋白酶自消化和较少的非特异性裂解，获得了更清洁的胰蛋白酶图谱。与传统的胰蛋白酶消化方法相比，胰蛋白酶消化的通量也有显著提高。(c)2009 Elsevier Inc. All rights reserved.
Trypsin digestion can induce artificial modifications Such as asparagine deamidation and N-terminal glutamine cyclization on proteins due to the temperature and the alkaline pH buffers used during digestion. The amount of these artificial modifications is directly proportional to the incubation time of protein samples in the reduction/alkylation buffer and, more important, in the digestion buffer where the peptides are completely solvent exposed. To minimize these artificial modifications, we focused oil minimizing the trypsin digestion time by maximizing trypsin activity. Trypsin activity was optimized by the complete removal of guanidine, which is a known trypsin inhibitor, from the digestion buffer. As a result, near complete trypsin digestion was achieved oil reduced and alkylated immunoglobulin gamma molecules in 30 min. The Protein tryptic fragments and their modification products were analyzed and quantified by reversed-phase liquid chromatography/tandem mass spectrometry using an in-line LTQ Orbitrap mass spectrometer. The reduction and alkylation reaction time was also minimized by monitoring the completeness of the reaction using a high-resolution time-of-flight mass spectrometer. Using this 30-min in-solution trypsin digestion method, little protocol-induced deamidation or N-terminal glutamine cyclization product was observed and cleaner tryptic maps were obtained due to less trypsin self-digestion and fewer nonspecific cleavages. The throughput of trypsin digestion was also improved significantly compared with conventional trypsin digestion methods. (c) 2009 Elsevier Inc. All rights reserved.