Protein fragmentation is a critical component of top-down proteomics, enabling gene-specific protein identification and full proteoform characterization. The factors that influence protein fragmentation include precursor charge, structure, and primary sequence, which have been explored extensively for collision-induced dissociation (CID). Recently, noticeable differences in CID-based fragmentation were reported for native versus denatured proteins, motivating the need for scoring metrics that are tailored specifically to native top-down mass spectrometry (nTDMS). To this end, position and intensity were tracked for 10,252 fragment ions produced by higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes. We used published structural data to explore the relationship between fragmentation and protein topology and revealed that fragmentation events occur at a large range of relative residue solvent accessibility. Additionally, our analysis found that fragment ions at sites with an N-terminal aspartic acid or a C-terminal proline make up on average 40 and 27%, respectively, of the total matched fragment ion intensity in nTDMS. Percent intensity contributed by each amino acid was determined and converted into weights to (1) update the previously published C-score and (2) construct a native Fragmentation Propensity Score. Both scoring systems showed an improvement in protein identification or characterization in comparison to traditional methods and overall increased confidence in results with fewer matched fragment ions but with high probability nTDMS fragmentation patterns. Given the rise of nTDMS as a tool for structural mass spectrometry, we forward these scoring metrics as new methods to enhance analysis of nTDMS data.

中文翻译：

---

使用 10,000 个片段离子为原生自上而下蛋白质组学的评分提供信息。

蛋白质片段化是自上而下蛋白质组学的关键组成部分，可实现基因特异性蛋白质鉴定和完整的蛋白质组表征。影响蛋白质断裂的因素包括前体电荷、结构和一级序列，这些因素已被广泛用于碰撞诱导解离 (CID)。最近，报告了天然蛋白质与变性蛋白质在基于 CID 的碎片化方面的显着差异，这促使需要专门为天然自上而下质谱 (nTDMS) 量身定制的评分指标。为此，对 159 种天然单体和 70 种复合物的高能碰撞解离 (HCD) 产生的 10,252 个碎片离子的位置和强度进行了跟踪。我们使用已发表的结构数据来探索片段化和蛋白质拓扑结构之间的关系，并揭示了片段化事件发生在大范围的相对残留溶剂可及性上。此外，我们的分析发现，N 端天冬氨酸或 C 端脯氨酸位点的碎片离子分别占 nTDMS 总匹配碎片离子强度的平均 40% 和 27%。确定每个氨基酸贡献的百分比强度并将其转换为权重以 (1) 更新先前公布的 C 分数和 (2) 构建一个天然的碎片倾向分数。与传统方法相比，两种评分系统都显示出蛋白质鉴定或表征的改进，并且总体上增加了对匹配碎片离子较少但具有高概率 nTDMS 碎片模式的结果的信心。鉴于 nTDMS 作为结构质谱分析工具的兴起，我们将这些评分指标作为增强 nTDMS 数据分析的新方法进行转发。