Top-down mass spectrometry of intact phosphorylated β-casein: Correlation between the precursor charge state and internal fragments
Top-down mass spectrometry of intact phosphorylated β-casein: Correlation between the precursor charge state and internal fragments
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DOI：
10.1002/jms.4364
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发表时间：
2019-06-01
影响因子：
2.3
通讯作者：
Green, Kari B.
中科院分区：
文献类型：
--
作者：
Chen, Jianzhong;Shiyanov, Pavel;Green, Kari B.
磷酸化蛋白在许多细胞过程中起着重要作用，相关磷蛋白的鉴定和表征有助于理解潜在的机制。在这里，我们报告了一个碰撞诱导解离自上而下的方法来表征磷蛋白的四极杆飞行时间质谱仪。β-酪蛋白是一种具有两种主要同种型和五个可磷酸化丝氨酸残基的蛋白质，被用作模型。检测到对应于电荷状态高达36（+）的完整β-酪蛋白离子的峰。对不同电荷状态（12（+）和15（+）至28（+））的β-酪蛋白离子进行串联质谱，以确定电荷状态对该蛋白解离的影响。大多数丰富的片段对应于y，B离子，和内部片段所造成的N-末端酰胺键附近的脯氨酸残基（XXX-Pro）的裂解。内部片段的丰度随着蛋白质前体离子的电荷状态而增加;这些内部片段主要来自一个或两个Xxx-Pro切割事件，并且难以准确分配。大量β-酪蛋白钠加合物的存在使光谱进一步复杂化。我们的研究结果表明，在解释磷蛋白和其他蛋白质的自上而下的质谱时，研究人员应该考虑内部片段和钠加合物的潜在形成，以进行可靠的表征。
Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision-induced dissociation top-down approach for characterizing phosphoproteins on a quadrupole time-of-flight mass spectrometer. beta-casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact beta-casein ions with charged states up to 36(+) were detected. Tandem mass spectrometry was performed on beta-casein ions of different charge states (12(+), and 15(+) to 28(+)) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N-terminal amide bond adjacent to proline residues (Xxx-Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx-Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of beta-casein further complicated the spectra. Our results suggest that when interpreting top-down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.