193 nm紫外光解作用下电荷减少对蛋白质序列覆盖的影响

IF 2.7 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Sean D. Dunham, , , Mohamed I. Gadallah, , and , Jennifer S. Brodbelt*, 
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引用次数: 0

摘要

自上而下质谱法的一个持续目标是提高对较大蛋白质的性能。使用更高能量的激活方法,如193nm紫外光解离(UVPD),提供了更广泛的大蛋白质碎片化的潜力,从而产生更大的序列覆盖。获得高序列覆盖率需要对片段离子进行可靠的识别和分配,而这一过程受到片段离子频谱拥塞和低信噪比(S/N)的阻碍。在这里,我们探索使用电荷还原方法来产生大蛋白质的低电荷状态,以增加离子积累,并产生片段离子的低电荷状态,这些片段离子自然分散在m/z域中,以减轻光谱拥挤。在烯醇化酶(47 kDa)和PRN-1 (63 kDa)的低荷电状态下,UVPD的序列覆盖率分别高达47%(烯醇化酶24+荷电状态)和23% (PRN-1 32+荷电状态),而标准高荷电状态下的序列覆盖率分别为17%(烯醇化酶55+荷电状态)和9% (PRN-1 55+荷电状态)。通过质子转移电荷还原反应(PTCR)进一步分散碎片离子在m/z域,增强识别。当在UVPD后使用PTCR时,烯醇酶(55+,67%)和PRN-1(55+, 34%)的最高电荷态的序列覆盖率最大,证实片段离子的电荷减少比前体离子的电荷减少对结果的影响更显着。通过结合电子转移高能碰撞解离(EThcD)和UVPD的结果(烯醇化酶的覆盖率为85%,PRN-1的覆盖率为52%),进一步提高了序列覆盖率,支持了互补MS/MS方法的使用,为自上而下分析较大蛋白质提供了更大的好处。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Impact of Charge Reduction on Sequence Coverage of Proteins by 193 nm Ultraviolet Photodissociation

Impact of Charge Reduction on Sequence Coverage of Proteins by 193 nm Ultraviolet Photodissociation

An ongoing goal of top-down mass spectrometry is to increase the performance for larger proteins. Using higher energy activation methods, like 193 nm ultraviolet photodissociation (UVPD), offers the potential to cause more extensive fragmentation of large proteins and thereby yield greater sequence coverage. Obtaining high sequence coverage requires confident identification and assignment of fragment ions, and this process is hampered by spectral congestion and low signal-to-noise ratio (S/N) of the fragment ions. Here we explore the use of charge reduction methods to produce lower charge states of large proteins to increase ion accumulation and generate lower charge states of fragment ions, ones that are naturally dispersed in the m/z domain to alleviate spectral congestion. UVPD of low charge states of enolase (47 kDa) and PRN-1 (63 kDa) resulted in sequence coverages as high as 47% (24+ charge state of enolase) and 23% (32+ of PRN-1) in comparison to 17% (55+ charge state of enolase) and 9% (55+ charge state of PRN-1) obtained for standard high charge states. Proton transfer charge reduction (PTCR) reactions were performed to further disperse fragment ions in the m/z domain and enhance identification. When employing PTCR after UVPD, sequence coverage was maximized for the highest charge states for enolase (55+, 67%) and PRN-1 (55+, 34%), confirming that charge reduction of fragment ions had a more notable impact on outcomes than charge reduction of precursor ions. Sequence coverages were increased further by combining results from electron transfer higher energy collision dissociation (EThcD) and UVPD (85% coverage for enolase and 52% coverage for PRN-1), bolstering the use of complementary MS/MS methods to yield greater dividends for top-down analysis of larger proteins.

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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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