Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma
Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma
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DOI：
10.1074/mcp.m800540-mcp200
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发表时间：
2009-08-01
影响因子：
7
通讯作者：
Borchers, Christoph H.
中科院分区：
文献类型：
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作者：
Kuzyk, Michael A.;Smith, Derek;Borchers, Christoph H.
基于质谱的多反应监测（MRM）蛋白质定量可以通过临床样品的快速、靶向、多重蛋白质表达谱分析显著影响生物标志物的发现和定量。创建了45种肽标准品的混合物，易于适应常见的血浆蛋白质组学工作流程，以允许绝对定量人血浆胰蛋白酶促蛋白酶中的45种内源性蛋白质。所有实验都是在人EDTA-血浆的简单胰蛋白酶消化物上进行的，没有预先的亲和力耗尽或富集。在胰蛋白酶消化后立即加入稳定的同位素标记的标准肽，因为发现在胰蛋白酶消化前加入稳定的同位素标记的标准肽会产生升高的和不可预测的结果。对所有45种蛋白质合成了含有同位素编码氨基酸（[C-13（6）]Arg或[C-13（6）]Lys）的蛋白质型胰蛋白酶肽。通过毛细管区带电泳评估肽纯度，并通过氨基酸分析确定肽量。为了获得最大的灵敏度和特异性，根据经验确定仪器参数以产生最丰富的前体离子和γ离子碎片。优化混合物中单个肽标准品的浓度，以接近分析物的内源性浓度，并确保MRM测定的最大线性动态范围。45种蛋白质中有43种在阿托摩尔水平的定量限下获得了良好的线性响应（r > 0.99）。
Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([C-13(6)]Arg or [C-13(6)]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (