Robustness and Ruggedness of Isoelectric Focusing and Superficially Porous Liquid Chromatography with Fourier Transform Mass Spectrometry.
Robustness and Ruggedness of Isoelectric Focusing and Superficially Porous Liquid Chromatography with Fourier Transform Mass Spectrometry.
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DOI：
10.1021/jasms.0c00355
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发表时间：
2021-01-06
影响因子：
3.2
通讯作者：
Patrie, Steven M.
中科院分区：
文献类型：
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作者：
Corbett, John R.;Robinson, Dana E.;Patrie, Steven M.
关键词：
完成了对由离胶等电聚焦 (IEF) 和表面多孔液相色谱 (SPLC) 与傅里叶变换质谱 (FTMS) 组成的多维蛋白质组学工作流程的研究，以评估与完整蛋白质测量相关的各种品质因数。以高和低 FTMS 分辨率对大肠杆菌蛋白质组进行三次重复分析，产生约 900 个 3 至 95 kDa 的冗余蛋白质形式。将色谱轴归一化为已识别的蛋白质形式，可以在蛋白质组重复之间进行可重复的理化性质测量，重复间方差为：<30 kDa 的蛋白质形式 ±3 ppm 质量误差、> 30 kDa 的蛋白质 ±1.1 Da、±12 s 保留时间误差和 ±0.21 pI 单位。大肠杆菌和标准蛋白的结果揭示了 pI 精度和蛋白质丰度之间的相关性，在多个 IEF 级分中检测到的物种的 pI 精度低于凝胶外系统的理论分辨率（分别为 ±0.05 与 ±0.17）。对标准蛋白质差异修饰的蛋白质形式的评估表明，高样品负载（100 微克）会改变 IEF pH 梯度曲线，导致样品变宽，从而有利于带电翻译后修饰（例如磷酸化、唾液酸化）的解析。尽管样品负载对 IEF 分辨率有影响，但直接测量或掺入大肠杆菌后测量的标准蛋白质的结果表明，工作流程的重现性允许跨 IEF 组分的 MS 信号重组，支持对 102-103 个样品量的完整蛋白质研究（蛋白质形式、蛋白质形式比率和蛋白质）进行三种无标记定量指标的评估，且检测限较低。
An investigation of a multidimensional proteomics workflow composed of off-gel isoelectric focusing (IEF) and superficially porous liquid chromatography (SPLC) with Fourier transform mass spectrometry (FTMS) was completed in order to assess various figures of merit associated with intact protein measurements. Triplicate analysis performed at both high and low FTMS resolutions on the E. coli proteome resulted in ~900 redundant proteoforms from 3 to 95 kDa. Normalization of the chromatographic axis to identified proteoforms enabled reproducible physicochemical property measurements between proteome replicates with inter-replicate variances of ±3 ppm mass error for proteoforms <30 kDa, ±1.1 Da for proteins >30 kDa, ±12 s retention time error, and ±0.21 pI units. The results for E. coli and standard proteins revealed a correlation between pI precision and proteoform abundance with species detected in multiple IEF fractions exhibiting pI precisions less than the theoretical resolution of the off-gel system (±0.05 vs ±0.17, respectively). Evaluation of differentially modified proteoforms of standard proteins revealed that high sample loads (100s μgrams) change the IEF pH gradient profile, leading to sample broadening that facilitates resolution of charged post-translational modifications (e.g., phosphorylation, sialylation). Despite the impact of sample load on IEF resolution, results on standard proteins measured directly or after being spiked into E. coli demonstrated that the reproducibility of the workflow permitted recombination of the MS signal across IEF fractions in a manner supporting the evaluation of three label-free quantitation metrics for intact protein studies (proteoforms, proteoform ratios, and protein) over 102–103 sample amount with low femtomole detection limits.
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影响因子：
2.9
作者：
BJELLQVIST, B;BASSE, B;CELIS, JE
通讯作者：
CELIS, JE
影响因子：
3.4
作者：
Kleinert, Peter;Kuster, Thomas;Troxler, Heinz
通讯作者：
Troxler, Heinz
影响因子：
2.9
作者：
CANTRELL, SJ;BABITCH, JA;TORRES, S
通讯作者：
TORRES, S
DOI：
10.1007/978-1-4939-3524-6_8
发表时间：
2016
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Ntai I;Toby TK;LeDuc RD;Kelleher NL
通讯作者：
Kelleher NL
影响因子：
64.8
作者：
通讯作者：
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