Chen Jianzhong, Shiyanov Pavel, Green Kari B
Department of Optometry and Vision Science, University of Alabama at Birmingham, Birmingham, Alabama, 35294.
Applied Biotechnology Branch, Air Force Research Laboratory, Dayton, Ohio, 45433.
J Mass Spectrom. 2019 Jun;54(6):527-539. doi: 10.1002/jms.4364.
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. β-casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact β-casein ions with charged states up to 36 were detected. Tandem mass spectrometry was performed on β-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 β-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.
磷酸化蛋白在许多细胞过程中发挥着重要作用，鉴定和表征相关的磷酸化蛋白有助于理解其潜在机制。在此，我们报道了一种在四极杆飞行时间质谱仪上用于表征磷酸化蛋白的碰撞诱导解离自上而下的方法。β-酪蛋白是一种具有两种主要异构体和五个可磷酸化丝氨酸残基的蛋白质，被用作模型。检测到了对应于电荷态高达36的完整β-酪蛋白离子的峰。对不同电荷态（12以及15至28）的β-酪蛋白离子进行串联质谱分析，以确定电荷态对该蛋白解离的影响。大多数丰富的碎片对应于y、b离子以及由与脯氨酸残基（Xxx-Pro）相邻的N端酰胺键断裂产生的内部碎片。内部碎片的丰度随着蛋白质前体离子的电荷态增加而增加；这些内部碎片主要来自一两个Xxx-Pro裂解事件，并且难以准确归属。β-酪蛋白大量钠加合物的存在使光谱进一步复杂化。我们的结果表明，在解释磷酸化蛋白和其他蛋白的自上而下质谱图时，研究人员应考虑内部碎片和钠加合物的潜在形成，以进行可靠的表征。
