A rapid, reproducible, on-the-fly orthogonal array optimization method for targeted protein quantification by LC/MS and its application for accurate and sensitive quantification of carbonyl reductases in human liver.
A rapid, reproducible, on-the-fly orthogonal array optimization method for targeted protein quantification by LC/MS and its application for accurate and sensitive quantification of carbonyl reductases in human liver.
复制标题
DOI：
10.1021/ac902314m
复制
发表时间：
2010-04-01
影响因子：
7.4
通讯作者：
Blanco, Javier G.
中科院分区：
文献类型：
--
作者：
Cao, Jin;Covarrubias, Vanessa M.;Straubinger, Robert M.;Wang, Hao;Duan, Xiaotao;Yu, Haoying;Qu, Jun;Blanco, Javier G.
选择反应监测（SRM）模式下的液相色谱（LC）/质谱（MS）为靶向蛋白定量提供了强有力的工具。然而，有效的，高通量的策略，适当选择的签名肽（SP）的蛋白质定量和准确优化其SRM条件仍然难以捉摸。在这里，我们描述了一种实时的正交阵列优化（OAO）方法，该方法可以在单个纳米流LC/MS运行中快速，全面和可重现地优化大量候选肽。在优化的条件下，许多肽候选人可以在生物基质中进行评估，以选择最终的SP。OAO策略采用系统的实验设计，在25个连续SRM试验的循环中策略性地改变产物离子，去簇能和碰撞能，这准确地揭示了这些因素对候选肽的信噪比的影响，并优化了每个因素。作为概念验证，我们开发了一种高度灵敏、准确且可重复的方法来定量人肝脏中的羰基还原酶CBR 1和CBR 3。通过nano-LC/LTQ/Orbitrap鉴定候选肽，使用一组严格的标准过滤，并进行OAO。在评估候选物的灵敏度和稳定性之后，选择两种SP用于定量每种蛋白质。由于测定条件的准确OAO，CBR 1和CBR 3的灵敏度分别达到80和110阿莫尔。该方法已被验证，并用于定量33人肝脏样品中的CBR。CBR 1的平均水平为总蛋白的93.4±49.7（范围：26.2-241）ppm，CBR 3为7.69±4.38（范围：1.26-17.9）ppm。本研究的关键观察结果是：i）评价靶基质中的肽稳定性对于最终选择SP是必不可少的; ii）利用两种独特的SP有助于靶蛋白定量的高可靠性;以及iii）使用经验证浓度的标准蛋白构建校准曲线是有益的，以避免如果单独使用合成肽可能导致的严重偏倚。总的来说，OAO方法是通用的，适用于高通量定量蛋白质组学发现实验鉴定的经验证的生物标志物。
Liquid chromatography (LC)/mass spectrometry (MS) in selected-reactions-monitoring (SRM) mode provides a powerful tool for targeted protein quantification. However, efficient, high-throughput strategies for proper selection of signature peptides (SP) for protein quantification and accurate optimization of their SRM conditions remain elusive. Here we describe an on-the-fly, orthogonal array optimization (OAO) approach that enables rapid, comprehensive, and reproducible SRM optimization of a large number of candidate peptides in a single nanoflow-LC/MS run. With the optimized conditions, many peptide candidates can be evaluated in biological matrices for selection of the final SP. The OAO strategy employs a systematic experimental design that strategically varies product ions, de-clustering energy and collision energy in a cycle of 25 consecutive SRM trials, which accurately reveals the effects of these factors on the single-to-noise ratio of a candidate peptide, and optimizes each. As proof of concept, we developed a highly sensitive, accurate, and reproducible method for the quantification of carbonyl reductases CBR1 and CBR3 in human liver. Candidate peptides were identified by nano-LC/LTQ/Orbitrap, filtered using a stringent set of criteria, and subjected to OAO. After evaluating both sensitivity and stability of the candidates, two SP were selected for quantification of each protein. As a result of the accurate OAO of assay conditions, sensitivities of 80 and 110 amol were achieved for CBR1 and CBR3, respectively. The method was validated and used to quantify the CBRs in 33 human liver samples. The mean level of CBR1 was 93.4±49.7 (range: 26.2–241) ppm of total protein, and for CBR3 was 7.69±4.38 (range: 1.26–17.9) ppm. Key observations of this study are that: i) evaluation of peptide stability in the target matrix is essential for final selection of the SP; ii) utilization of two unique SP contributes to high reliability of target protein quantification; and iii) it is beneficial to construct calibration curves using standard proteins of verified concentrations to avoid severe biases that may result if synthesized peptides alone are used. Overall, the OAO method is versatile and adaptable to high-throughput quantification of validated biomarkers identified by proteomic discovery experiments.
登录
查看更多内容
影响因子：
4.4
作者：
Barnidge, DR;Goodmanson, MK;Muddiman, DC
通讯作者：
Muddiman, DC
影响因子：
4.4
作者：
Anderson, NL;Anderson, NG;Pearson, TW
通讯作者：
Pearson, TW
影响因子：
3.9
作者：
Kassner, Nina;Huse, Klaus;Wojnowski, Leszek
通讯作者：
Wojnowski, Leszek
影响因子：
4.4
作者：
Duan X;Young R;Straubinger RM;Page B;Cao J;Wang H;Yu H;Canty JM;Qu J
通讯作者：
Qu J
DOI：
10.1074/mcp.m700504-mcp200
发表时间：
2008-11
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Martin DB;Holzman T;May D;Peterson A;Eastham A;Eng J;McIntosh M
通讯作者：
McIntosh M