Quantitative Bottom-Up Proteomics Depends on Digestion Conditions
Quantitative Bottom-Up Proteomics Depends on Digestion Conditions
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DOI：
10.1021/ac4027274
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发表时间：
2014-01-07
影响因子：
7.4
通讯作者：
Stein, Stephen E.
中科院分区：
文献类型：
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作者：
Lowenthal, Mark S.;Liang, Yuxue;Stein, Stephen E.
准确的定量是蛋白质组学和生物标记物发现领域以及临床诊断分析的基本要求。为了证明由于“典型”蛋白质消化方法的不同，可测量的多肽浓度的定量变化程度，使用12种不同的样品制备方法对模型蛋白质人血清白蛋白(HSA)进行了酶消化，并通过全面的胰酶分解时间过程分别进行了检测。探讨了不同的消化条件，包括消化时间、变性剂、酶的来源、样品清理和变性温度等方面的差异。时间过程实验比较了从15分钟到48小时胰酶消化的相对多肽浓度的差异。在LC-MS分析之前，将在酵母中表达的简化的稳定同位素标记的全长(HSA)蛋白(N-15形式)添加到所有样品中，以比较多种胰蛋白酶多肽的产量。在三重四极杆(QQQ)质谱仪上进行多反应监测(MRM)，用高效液相色谱-串联质谱仪(LCMS/MS)对积分提取离子色谱图(XIC)进行归一化，实现相对定量，并监测相关的多肽碎片转变和多个多肽电荷状态，以验证定量结果。结果表明，对于大多数多肽，包括所谓的“蛋白质型”多肽，蛋白质浓度与胰蛋白酶多肽浓度不相等。在消化过程中，肽的释放表现出复杂的动力学，取决于消化条件，由此推断，还取决于变性蛋白质的结构。胰酶裂解部位的水解率也受到最近氨基酸残基和次近氨基酸残基差异的影响。这些数据表明了酶蛋白消化的非化学计量，强调了常规绝对蛋白质定量经常被忽视的困难，并强调了使用适当的内标和同位素稀释技术的必要性。
Accurate quantification is a fundamental requirement in the fields of proteomics and biomarker discovery, and for clinical diagnostic assays. To demonstrate the extent of quantitative variability in measurable peptide concentrations due to differences among "typical" protein digestion protocols, the model protein, human serum albumin (HSA), was subjected to enzymatic digestion using 12 different sample preparation methods, and separately, was examined through a comprehensive timecourse of trypsinolysis. A variety of digestion conditions were explored including differences in digestion time, denaturant, source of enzyme, sample cleanup, and denaturation temperature, among others. Timecourse experiments compared differences in relative peptide concentrations for tryptic digestions ranging from 15 min to 48 h. A predigested stable isotope-labeled (N-15 form of the full-length (HSA) protein, expressed in yeast was spiked into all samples prior to LC-MS analysis to compare yields of numerous varieties of tryptic peptides. Relative quantification was achieved by normalization of integrated extracted ion chromatograms (XICs) using liquid chromatography-tandem mass spectrometry (LCMS/MS) by multiple-reaction monitoring (MRM) on a triple quadrupole (QQQ) MS. Related peptide fragmentation transitions, and multiple peptide charge states, were monitored for validation of quantitative results. Results demonstrate that protein concentration was shown to be unequal to tryptic peptide concentrations for most peptides, including so-called "proteotypic" peptides. Peptide release during digestion displayed complex kinetics dependent on digestion conditions and, by inference, from denatured protein structure. Hydrolysis rates at tryptic cleavage sites were also shown to be affected by differences in nearest and next-nearest amino acid residues. The data suggesting nonstoichiometry of enzymatic protein digestions emphasizes the often overlooked difficulties for routine absolute protein quantification, and highlights the need for use of suitable internal standards and isotope dilution techniques.