A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin.
A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin.
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DOI：
10.1021/pr100656u
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发表时间：
2010-10-01
影响因子：
4.4
通讯作者：
Borchers CH
中科院分区：
文献类型：
--
作者：
Proc JL;Kuzyk MA;Hardie DB;Yang J;Smith DS;Jackson AM;Parker CE;Borchers CH
血浆生物标志物研究基于不同治疗组之间或患病人群与对照人群之间的蛋白质差异表达。然而，大多数基于质谱的蛋白质定量方法是基于肽的检测和定量，而不是完整的蛋白质。为了使基于肽的蛋白质定量准确，蛋白质组学分析中使用的消化方案必须是有效的和可重复的。然而，很少有研究使用绝对定量方法比较血浆变性/消化方案。在本文中，14个组合的热，溶剂[乙腈，甲醇，三氟乙醇]，离液剂[盐酸胍，尿素]，和表面活性剂[十二烷基硫酸钠（SDS）和脱氧胆酸钠（DOC）]进行了比较，就其有效性，提高随后的胰蛋白酶消化。这些消化方案进行了评价，通过定量生产的蛋白质型胰蛋白酶肽从45中等至高丰度的血浆蛋白，使用串联质谱在多反应监测模式，与稳定同位素标记的类似物的混合物，这些蛋白质型肽作为内标。当比较这14种方法的消化效率时，我们发现，与更常用的尿素方案相比，两种表面活性剂（SDS和DOC）都使来自这45种蛋白质的胰蛋白酶肽的总产率增加。然而，SDS可能对随后的质谱分析造成严重干扰。DOC，另一方面，可以很容易地从样品中除去的酸沉淀。检查重复性研究的结果，用5个重复的消化时间进行9小时的消化，DOC和SDS产生最高的平均消化效率（~80%），具有最高的平均重复性（<5%误差，定义为与平均值的相对偏差）。然而，由于使用SDS可能产生干扰，我们建议DOC与9小时消化程序作为最佳方案。
Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides from 45 moderate- to high-abundance plasma proteins, using tandem mass spectrometry in multiple reaction monitoring mode, with a mixture of stable-isotope labeled analogues of these proteotypic peptides as internal standards. When the digestion efficiencies of these 14 methods were compared, we found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol. SDS, however, can be a serious interference for subsequent mass spectrometry. DOC, on the other hand, can be easily removed from the samples by acid precipitation. Examining the results of a reproducibility study, done with 5 replicate digestions, DOC and SDS with a 9 h digestion time produced the highest average digestion efficiencies (~80%), with the highest average reproducibility (<5% error, defined as the relative deviation from the mean value). However, because of potential interferences resulting from the use of SDS, we recommend DOC with a 9 h digestion procedure as the optimum protocol.
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