Nonisotopic Reagents for a Cost-Effective Increase in Sample Throughput of Targeted Quantitative Proteomics

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• 作者：Mary Joan Castillo ; Adam J. McShane ; Min Cai ; Yuanyuan Shen ; Lei Wang ; Xudong Yao
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：September 15, 2015
• 年：2015
• 卷：87
• 期：18
• 页码：9209-9216
• 全文大小：340K
• ISSN：1520-6882

文摘

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.