Development of a highly automated and multiplexed targeted proteome pipeline and assay for 112 rat brain synaptic proteins.
Development of a highly automated and multiplexed targeted proteome pipeline and assay for 112 rat brain synaptic proteins.
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DOI：
10.1002/pmic.201400353
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发表时间：
2015-04
期刊：
影响因子：
3.4
通讯作者：
Williams KR
中科院分区：
文献类型：
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作者：
Colangelo CM;Ivosev G;Chung L;Abbott T;Shifman M;Sakaue F;Cox D;Kitchen RR;Burton L;Tate SA;Gulcicek E;Bonner R;Rinehart J;Nairn AC;Williams KR
我们为大规模（>1000 次离子对/运行）无标记 LC-MRM 蛋白质组检测提供了全面的工作流程。创新包括自动 MRM 离子对选择、可将信号/噪声提高 2 倍以上的智能保留时间安排 (xMRM) 以及自动峰值建模。数据分析的改进包括新颖的 Q/C 指标、归一化组面积比 (NGAR)、MLR 归一化、加权回归分析以及通过耶鲁蛋白质表达数据库进行数据传播。作为原理证明，我们针对小鼠/大鼠突触后密度 (PSD) 组分开发了一种强大的 90 分钟 LC-MRM 测定法，基于每种蛋白质 15 次观察，对 112 种蛋白质中的 337 种肽进行常规定量。使用稳定同位素稀释肽标准品 (SIS) 进行的平行分析表明，无标记分析和 SIS 分析之间的保留时间 (1.0) 和蛋白质倍数变化 (0.94) 具有非常高的相关性。总体而言，我们的第一种方法实现了 11.4% 的技术 CV，在无需用户干预的情况下对 1697 个离子对中的 97.5% 进行了定量，从而实现了高效、稳健的单次进样 LC-MRM 测定。
We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling (xMRM) that improves Signal/Noise by >2-fold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, Normalized Group Area Ratio (NGAR), MLR normalization, weighted regression analysis, and data dissemination through the Yale Protein Expression Database. As a proof of principle we developed a robust 90 minute LC-MRM assay for Mouse/Rat Post-Synaptic Density (PSD) fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our first method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay.