Enhancing Top-Down Analysis of Proteins by Combining Ultraviolet Photodissociation (UVPD), Proton-Transfer Charge Reduction (PTCR), and Gas-Phase Fractionation to Alleviate the Impact of Nondissociated Precursor Ions
Enhancing Top-Down Analysis of Proteins by Combining Ultraviolet Photodissociation (UVPD), Proton-Transfer Charge Reduction (PTCR), and Gas-Phase Fractionation to Alleviate the Impact of Nondissociated Precursor Ions
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DOI：
10.1021/jasms.3c00351
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发表时间：
2023-12-27
影响因子：
3.2
通讯作者：
Brodbelt，Jennifer S.
中科院分区：
文献类型：
--
作者：
Dunham，Sean D.;Brodbelt，Jennifer S.
自顶向下质谱分析策略的最新进展继续改善完整蛋白质的分析。193nm紫外光解离（UVPD）是一种适合于自顶向下分析的方法。UVPD通常使用相对较低的光子通量进行，以限制片段离子的多代解离并最大化序列覆盖。因此，前体离子的很大一部分在UVPD过程中幸存下来，在光谱中占主导地位，并可能阻碍碎片离子的识别。在这里，我们探索在UVPD后分离低于和高于前体离子的片段离子亚群，作为消除幸存的前体离子对检测低丰度片段离子的影响的一种手段。这种气相分馏方法提高了片段离子的序列覆盖率，这些片段离子位于比前驱体低和高的区域，平均分别提高了1.3和2.3倍。将这种气相分馏方法与质子转移电荷还原（PTCR）相结合，进一步提高了em/zregions的序列覆盖率，分别提高了1.3和1.4倍。采用6个分离事件的后UVPD分离+ PTCR策略，烯醇化酶的序列覆盖率为75%，这是193 nm UVPD中最高的。
Recent advances in top-down mass spectrometry strategies continue to improve the analysis of intact proteins. 193 nm ultraviolet photodissociation (UVPD) is one method well-suited for top-down analysis. UVPD is often performed using relatively low photon flux in order to limit multiple-generation dissociation of fragment ions and maximize sequence coverage. Consequently, a large portion of the precursor ion survives the UVPD process, dominates the spectrum, and may impede identification of fragment ions. Here, we explore the isolation of subpopulations of fragment ions lower and higher than the precursor ion after UVPD as a means to eliminate the impact of the surviving precursor ion on the detection of low abundance fragment ions. This gas-phase fractionation method improved sequence coverage harvested from fragment ions found in them/zregions lower and higher than the precursor by an average factor of 1.3 and 2.3, respectively. Combining this gas-phase fractionation method with proton transfer charge reduction (PTCR) further increased the sequence coverage obtained from thesem/zregions by another factor of 1.3 and 1.4, respectively. Implementing a post-UVPD fractionation + PTCR strategy with six fractionation events resulted in a sequence coverage of 75% for enolase, the highest reported for 193 nm UVPD.