Department of Chemistry, University of Texas, Austin, Texas 78712, United States.
J Am Soc Mass Spectrom. 2022 Mar 2;33(3):446-456. doi: 10.1021/jasms.1c00296. Epub 2022 Feb 4.
Ultraviolet photodissociation (UVPD) mass spectrometry has gained attention in recent years for its ability to provide high sequence coverage of intact proteins. However, secondary dissociation of fragment ions, in which fragment ions subjected to multiple laser pulses decompose into small products, is a common phenomenon during UVPD that contributes to limited coverage in the midsection of protein sequences. To counter secondary dissociation, a method involving the application of notched waveforms to modulate the trajectories of fragment ions away from the laser beam, termed fragment ion protection (FIP), was previously developed to reduce the probability of secondary dissociation. This, in turn, increased the number of identified large fragment ions. In the present study, FIP was applied to UVPD of large proteins ranging in size from 29 to 55 kDa, enhancing the abundances of large fragment ions. A stepped-FIP strategy was implemented in which UVPD mass spectra were collected using multiple different amplitudes of the FIP waveforms and then the results from the mass spectra were combined. By using stepped-FIP, the number of fragment ions in the midsections of the sequences increased for all proteins. For example, whereas no fragment ions were identified in the middle section of the sequence for glutamate dehydrogenase (55 kDa, 55+ charge state), 10 sequence ions were identified by using UVPD-FIP.
紫外光解吸（UVPD）质谱法近年来因其能够提供完整蛋白质的高序列覆盖率而受到关注。然而，在 UVPD 过程中，片段离子的二次解离（其中，经受多次激光脉冲的片段离子分解成小产物）是一种常见现象，这导致蛋白质序列中间部分的覆盖度有限。为了应对二次解离，先前开发了一种涉及应用缺口波形来调节片段离子远离激光束的轨迹的方法，称为片段离子保护（FIP），以降低二次解离的概率。这反过来又增加了大片段离子的数量。在本研究中，FIP 应用于大小为 29 至 55 kDa 的大型蛋白质的 UVPD，增强了大片段离子的丰度。采用逐步 FIP 策略，使用多个不同幅度的 FIP 波形收集 UVPD 质谱，并将质谱结果进行组合。通过使用逐步 FIP，所有蛋白质的序列中间部分的片段离子数量都增加了。例如，谷氨酸脱氢酶（55 kDa，55+电荷态）的序列中间部分没有鉴定出片段离子，但通过使用 UVPD-FIP 鉴定出了 10 个序列离子。
