High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer
High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer
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DOI：
10.1021/acs.jproteome.7b00043
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发表时间：
2017-05-01
影响因子：
4.4
通讯作者：
Brodbelt, Jennifer S.
中科院分区：
文献类型：
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作者：
Cleland, Timothy P.;DeHart, Caroline J.;Brodbelt, Jennifer S.
通过质谱分析完整蛋白质（自上而下的策略）具有阐明蛋白质形态变异的巨大潜力，包括翻译后修饰（PTMs）的模式，这可能无法通过单独分析肽（自下而上的方法）来识别。为了最大限度地扩大PTMs的序列覆盖范围和定位，人们开发了各种片段化模式，从完整蛋白质的深处产生片段离子。紫外光解（UVPD）最近被证明可以在多种蛋白质上产生高序列覆盖率和PTM保留，并且在色谱时间尺度上具有越来越多的有效性证据。然而，迄今为止，利用UVPD进行高通量自上而下的分析受到生物信息学的限制。本研究采用定性自顶向下蛋白质组学方法对HeLa全细胞裂解液进行比较分析，UVPD检测153个蛋白和489个蛋白形态，高能碰撞解离（HCD）检测271个蛋白和982个蛋白形态。在检测到的全部蛋白质形态中，有286种在UVPD和HCD数据集之间存在重叠，其中68%的蛋白质形态在UVPD和HCD中的C分大于40分。发现UVPD的平均序列覆盖率（UVPD为28.20%，HCD为17.8%,p < 0.0001）高于HCD，并且UVPD数据集的q值有改善的趋势。该研究证明了UVPD和HCD在更广泛的蛋白质分析和蛋白质形态表征方面的互补性。
The analysis of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by analysis of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. Ultraviolet photodissociation (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatographic time scale. However, utilization of UVPD for high-throughput top-down analysis to date has been limited by bioinformatics. Here we detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher energy collisional dissociation (HCD) in a comparative analysis of HeLa whole-cell lysate by qualitative top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD data sets, with 68% of proteoforms having C scores greater than 40 for UVPD and 63% for HCD. The average sequence coverage (28 20% for UVPD versus 17 8% for HCD, p < 0.0001) was found to be higher for UVPD than HCD and with a trend toward improvement in q value for the UVPD data set. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.