Grimaud Arthur, Babović Maša, Holck Frederik Haugaard, Jensen Ole N, Schwämmle Veit
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Mol Cell Proteomics. 2025 May;24(5):100896. doi: 10.1016/j.mcpro.2024.100896. Epub 2025 Feb 13.
Tandem mass spectrometry of peptides and proteins generates 3mass spectra of their gas-phase fragmentation product ions, including N-terminal, C-terminal, and internal fragment ions. While N- and C-terminal ions are routinely assigned and identified using computational methods, internal fragment ions are often difficult to annotate correctly. They become particularly relevant for long peptides and full proteoforms where the peptide backbone is more likely to be fragmented multiple times. Internal fragment ions potentially offer tremendous information regarding amino acid sequences and positions of post-translational modifications of peptides and intact proteins. However, their practical application is challenged by the vast number of theoretical internal fragments that exist for long amino acid sequences, leading to a high risk of false-positive annotations. We analyze the mass spectral contributions of internal fragment ions in spectra from middle-down and top-down experiments and introduce a novel graph-based annotation approach designed to manage the complexity of internal fragments. Our graph-based representation allows us to compare multiple candidate proteoforms in a single graph, and to assess different candidate annotations in a fragment ion spectrum. We demonstrate cases from middle-down and top-down data where internal ions enhance amino acid sequence coverage of polypeptides and proteins and accurate localization of post-translational modifications. We conclude that our graph-based method provides a general approach to process complex tandem mass spectra, enhance annotation of internal fragment ions, and improve proteoform sequencing and characterization by mass spectrometry.
肽和蛋白质的串联质谱分析会生成其气相碎裂产物离子的质谱图，包括N端、C端和内部碎片离子。虽然N端和C端离子通常使用计算方法进行归属和鉴定，但内部碎片离子往往难以正确注释。对于长肽和完整蛋白质形式而言，内部碎片离子尤为重要，因为肽主链更有可能多次碎裂。内部碎片离子可能提供有关肽和完整蛋白质的氨基酸序列以及翻译后修饰位置的大量信息。然而，它们的实际应用受到长氨基酸序列存在的大量理论内部碎片的挑战，导致假阳性注释的风险很高。我们分析了中向下和自上而下实验光谱中内部碎片离子的质谱贡献，并引入了一种新颖的基于图的注释方法，旨在管理内部碎片的复杂性。我们基于图的表示使我们能够在单个图中比较多个候选蛋白质形式，并评估碎片离子光谱中的不同候选注释。我们展示了中向下和自上而下数据中的案例，其中内部离子增强了多肽和蛋白质的氨基酸序列覆盖范围以及翻译后修饰的准确定位。我们得出结论，我们基于图的方法提供了一种处理复杂串联质谱图、增强内部碎片离子注释以及通过质谱改进蛋白质形式测序和表征的通用方法。
