Optimization of digestion parameters for protein quantification
Optimization of digestion parameters for protein quantification
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DOI：
10.1016/j.ab.2009.05.050
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发表时间：
2009-10-01
影响因子：
2.9
通讯作者：
Barr, John R.
中科院分区：
文献类型：
--
作者：
Norrgran, Jessica;Williams, Tracie L.;Barr, John R.
我们提出了一种快速有效的溶液中酶消化方案，适用于基于质谱的绝对蛋白质定量技术。消化方法采用RapiGest SF（一种酸不稳定表面活性剂）、过量的改性胰蛋白酶（酶与底物的比例为2.5：1）和2 h的孵育时间。不使用还原/烷基化试剂。系统地改变消化参数，以监测其对消化速率和完全性的影响。为了证明该方法的普遍适用性，使用病毒血凝素（HA）作为模型蛋白进行优化，然后应用于蓖麻毒素，一种从蓖麻子（蓖麻）中提取的有效蛋白质毒素。优化的参数包括孵育时间、RapiGest SF浓度、酶与底物的比例和孵育温度。通过比较来自HA蛋白两个不同位点的两种蛋白质特异性肽的产率来进行优化。通过液相色谱-串联质谱法在多反应监测模式下使用同位素标记的肽标准品进行定量分析。(C)2009年由Elsevier Inc.出版
We present a rapid and efficient in-solution enzymatic digestion protocol suitable for mass spectrometry-based absolute protein quantification techniques. The digestion method employs RapiGest SF (an acid-labile surfactant), an excess amount of modified trypsin (enzyme-to-substrate ratio of 2.5:1), and an incubation time of 2 h. No reduction/alkylation reagents are used. Digestion parameters were varied systematically to monitor their effect on rate and completeness of digestion. To demonstrate the general applicability of the method, the optimization was done using a viral hemagglutinin (HA) as a model protein and then applied to ricin, a potent protein toxin extracted from the castor bean (Ricinus communis). The parameters that were optimized included incubation time, concentration of RapiGest SF, enzyme-to-substrate ratio, and incubation temperature. The optimization was done by comparing the yields from two protein-specific peptides originating from two different sites of the HA protein. The analysis was performed by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode using isotopically labeled peptide standards for quantification. (C) 2009 Published by Elsevier Inc.