Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California 90095, United States.
Department of Chemistry, University of California Riverside, Riverside, California 92521, United States.
J Am Soc Mass Spectrom. 2021 Jul 7;32(7):1752-1758. doi: 10.1021/jasms.1c00113. Epub 2021 Jun 8.
Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N- or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photon-based fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome , myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15-20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.
自上而下的质谱分析（TD-MS）可对完整蛋白质进行分析，得到的片段离子可与蛋白质的一级序列相关联。生成的片段可以是包含 N 端或 C 端的末端片段，也可以是既不包含 N 端也不包含 C 端的内部片段。传统的 TD-MS 实验中，由于这些片段的分配存在歧义，内部片段的生成通常会被避免。在这里，我们证明在基于碰撞、基于电子和基于光子的碎片化方法中可以形成和分配内部片段，并且这些片段富含序列信息，可以解释更大程度的蛋白质一级序列。对于三种测试蛋白细胞色素、肌红蛋白和碳酸酐酶 II，在分析中纳入内部片段可使序列覆盖率增加约 15-20%，且序列覆盖率不少于 85%。包含末端片段和内部片段的分配可实现接近完整的蛋白质序列覆盖。因此，通过在 TD-MS 分析中同时包含末端和内部片段的分配，可以实现深度蛋白质序列分析，更可靠地定位修饰位点。
