193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes.
193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes.
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DOI：
10.1021/acs.jproteome.6b00289
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发表时间：
2016-08-05
影响因子：
4.4
通讯作者：
Brodbelt JS
中科院分区：
文献类型：
--
作者：
Robinson MR;Taliaferro JM;Dalby KN;Brodbelt JS
液相色谱串联质谱 (LC-MS/MS) 的进步使得大规模的磷酸化蛋白质组分析成为可能，但与可靠的磷酸盐定位相关的持续挑战继续推动新裂解技术的开发。在本研究中，评估了 193 nm 处的紫外光解离 (UVPD)，以表征正离子和负离子模式下的磷酸肽。与更标准的高能碰撞解离 (HCD) 相比，UVPD 提供了更广泛的裂解，并改善了产物中的磷酸盐保留。负模式 UVPD 在检测和测序来自 α 和 β 酪蛋白的高酸性磷酸肽方面显示出特别的优点，但由于负模式下的电离效率较低，因此对于大规模分析来说不那么稳健。通过 UVPD 和 HCD 分析 HeLa 和 HCC70 细胞裂解物。虽然与 UVPD 相比，HCD 识别出更多的磷酸肽和蛋白质，但与单独使用任一方法相比，UVPD 分析的独特匹配可以与 HCD 数据集相结合，以提高总体覆盖深度。
Advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) have permitted phosphoproteomic analysis on a grand scale, but ongoing challenges specifically associated with confident phosphate localization continue to motivate the development of new fragmentation techniques. In the present study, ultraviolet photodissociation (UVPD) at 193 nm is evaluated for the characterization of phosphopeptides in both positive and negative ion modes. Compared to the more standard higher energy collisional dissociation (HCD), UVPD provided more extensive fragmentation with improved phosphate retention on productions. Negative mode UVPD showed particular merit for detecting and sequencing highly acidic phosphopeptides from alpha and beta casein, but was not as robust for larger scale analysis due to lower ionization efficiencies in the negative mode. HeLa and HCC70 cell lysates were analyzed by both UVPD and HCD. While HCD identified more phosphopeptides and proteins compared to UVPD, the unique matches from UVPD analysis could be combined with the HCD data set to improve the overall depth of coverage compared to either method alone.