Nonisotopic reagents for a cost-effective increase in sample throughput of targeted quantitative proteomics.
Nonisotopic reagents for a cost-effective increase in sample throughput of targeted quantitative proteomics.
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DOI：
10.1021/acs.analchem.5b01727
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发表时间：
2015-08
影响因子：
7.4
通讯作者：
M. Castillo;A. McShane;Min-Min Cai-Min;Yuanyuan Shen;Lei Wang;Xudong Yao
中科院分区：
文献类型：
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作者：
M. Castillo;A. McShane;Min-Min Cai-Min;Yuanyuan Shen;Lei Wang;Xudong Yao
超通量质谱（uMS）新技术将质谱（MS）固有的分析物复用能力转化为样品复用能力。uMS的核心技术优势在于解耦使用同位素定量参考和样品的非同位素质量编码。这些优势包括：(1)高样品通量潜力；(2)利用少量昂贵的稳定同位素作为定量参考；(3)释放非同位素试剂化学结构多样性的开源探索，显著提高分析物的质谱可检测性。报道了一种特殊的uMS方法，超通量多反应监测（uMRM），用于在多个血清样本中对前列腺特异性抗原（PSA）的替代肽（SVILLGR）进行一次实验定量。在每个样品中对加标同位素参考（SVILLGR*）和内源性天然肽进行衍生化后，将所有样品汇集在一起，使用固定化抗体同时富集和清除衍生化肽对。合并样品的质谱分析报告了替代肽的数量和样品来源。提出了几种不同样品通量的分析，最高的是15-in-1。非同位素试剂的筛选使用多肽基化合物组合文库，试剂的选择基于多肽的衍生化有效性和质谱信号增强能力。发现uMRM质谱技术的精密度、准确度和线性度与标准同位素稀释MRM质谱相当。
The new technology of ultrathroughput MS (uMS) transforms the intrinsic capability of analyte multiplexing in mass spectrometry (MS) to sample multiplexing. Core technological advantages of uMS rely on the decoupled use of isotopic quantitation reference and nonisotopic mass coding of samples. These advantages include: (1) high sample-throughput potential, (2) utilization of minimal amounts of expensive stable isotopes for the quantitation reference, and (3) unleashing of the open-source exploration of the chemical structure diversity of nonisotopic reagents to significantly enhance the MS detectability of analytes. A particular uMS method, ultrathroughput multiple reaction monitoring (uMRM), is reported for one-experiment quantitation of a surrogate peptide (SVILLGR) of prostate specific antigen (PSA) in multiple serum samples. Following derivatization of the pair of spiked, isotopic reference (SVILLGR*) and endogenous, native peptide in each sample, all samples were pooled for a step of simultaneous enrichment and cleanup of derivatized peptide pairs using immobilized antibody. The MS analysis of the pooled sample reported the quantity and sample origin of the surrogate peptide. Several analyses with different sample throughput were presented, with the highest being 15-in-1. Screening of nonisotopic reagents used combinatorial libraries of peptidyl compounds, and the reagent selection was based on the derivatization effectiveness and the capability of MS signal enhancement for the peptide. The precision, accuracy, and linearity of the uMRM MS technology were found to be comparable with standard isotope dilution MRM MS.