Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
J Proteome Res. 2021 Dec 3;20(12):5412-5418. doi: 10.1021/acs.jproteome.1c00599. Epub 2021 Nov 5.
A large fraction of observed fragment ion intensity remains unidentified in top-down proteomics. The elucidation of these unknown fragment ions could enable researchers to identify additional proteoforms and reduce proteoform ambiguity in their analyses. Internal fragment ions have received considerable attention as a major source of these unidentified fragment ions. Internal fragments are product ions that contain neither protein terminus, in contrast with terminal ions that contain a single terminus. There are many more possible internal fragments than terminal fragments, and the resulting computational complexity has historically limited the application of internal fragment ions to low-complexity samples containing only one or a few proteins of interest. We implemented internal fragment ion functionality in MetaMorpheus to allow the proteome-wide annotation of internal fragment ions. MetaMorpheus first uses terminal fragment ions to identify putative proteoforms and then employs internal fragment ions to disambiguate similar proteoforms. In the analysis of mammalian cell lysates, we found that MetaMorpheus could disambiguate over half of its previously ambiguous proteoforms while also providing up to a 7% increase in proteoform-spectrum matches identified at a 1% false discovery rate.
在自上而下的蛋白质组学中，大量观察到的片段离子强度仍然无法识别。阐明这些未知的片段离子可以使研究人员能够识别更多的蛋白质异构体，并减少分析中的蛋白质异构体歧义。内部片段离子作为这些未知片段离子的主要来源受到了相当多的关注。内部片段是既不包含蛋白质末端的产物离子，与仅包含单个末端的末端离子相反。内部片段的可能性比末端片段多得多，历史上计算的复杂性限制了内部片段离子的应用，仅适用于包含一个或几个感兴趣的蛋白质的低复杂度样本。我们在 MetaMorpheus 中实现了内部片段离子功能，以允许对内部片段离子进行全蛋白质组注释。MetaMorpheus 首先使用末端片段离子来识别可能的蛋白质异构体，然后使用内部片段离子来消除相似的蛋白质异构体的歧义。在哺乳动物细胞裂解物的分析中，我们发现 MetaMorpheus 可以消除其之前超过一半的模糊蛋白质异构体，同时在 1%假发现率下还可以增加高达 7%的蛋白质谱匹配。
