Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry.
Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry.
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DOI：
10.1007/s13361-017-1635-x
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发表时间：
2017-06
影响因子：
3.2
通讯作者：
Kelleher NL
中科院分区：
文献类型：
--
作者：
Haverland NA;Skinner OS;Fellers RT;Tariq AA;Early BP;LeDuc RD;Fornelli L;Compton PD;Kelleher NL
完整蛋白质在气相中的断裂受氨基酸组成、前体离子的质量和电荷、高阶结构和使用的解离技术的影响。一对残基之间发生碎片的可能性称为碎片倾向性，通过将分配的碎片事件总数除以每个残基对可能的碎片事件总数来计算。在这里，我们描述了使用变性或原生电喷雾电离进行自上而下质谱（TDMS）时的一般碎片倾向。采用nTDMS （native - top-down mass spectrometry）对131种蛋白质形态进行了165次实验，共收集到5311个匹配的片段位点。这些数据用于确定399对残基的断裂倾向。与变性自顶向下质谱（dTDMS）相比，nTDMS中发生的n端到脯氨酸或c端到天冬氨酸的断裂途径比其他残基更强。更普遍的是，与使用dTDMS相比，使用nTDMS时，257/399（64%）的破碎倾向发生了显著变化（p≤0.05），其中123个发生了2倍或更大的变化。TDMS在天然模式下与变性模式相比，最显著的断裂倾向增强发生在(1)c -末端到天冬氨酸，(2)苯丙氨酸和色氨酸之间（F|W），(3)色氨酸和丙氨酸之间（W|A）。这里提出的碎片化倾向将在开发用于完整蛋白质和蛋白质复合物的nTDMS的定制评分系统中具有很高的价值。
Fragmentation of intact proteins in the gas phase is influenced by amino acid composition, the mass and charge of precursor ions, higher order structure, and the dissociation technique used. The likelihood of fragmentation occurring between a pair of residues is referred to as the fragmentation propensity and is calculated by dividing the total number of assigned fragmentation events by the total number of possible fragmentation events for each residue pair. Here, we describe general fragmentation propensities when performing top-down mass spectrometry (TDMS) using denaturing or native electrospray ionization. A total of 5,311 matched fragmentation sites were collected for 131 proteoforms that were analyzed over 165 experiments using native top-down mass spectrometry (nTDMS). These data were used to determine the fragmentation propensities for 399 residue pairs. In comparison to denatured top-down mass spectrometry (dTDMS), the fragmentation pathways occurring either N-terminal to proline or C-terminal to aspartic acid were even more enhanced in nTDMS as compared to other residues. More generally, 257/399 (64%) of the fragmentation propensities were significantly altered (p ≤0.05) when using nTDMS as compared to dTDMS and of these, 123 were altered by 2-fold or greater. The most notable enhancements of fragmentation propensities for TDMS in native vs. denatured mode occurred (1) C-terminal to aspartic acid, (2) between phenylalanine and tryptophan (F|W), and (3) between tryptophan and alanine (W|A). The fragmentation propensities presented here will be of high value in the development of tailored scoring systems used in nTDMS of both intact proteins and protein complexes.