Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections.
Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections.
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DOI：
10.1021/acs.analchem.1c05171
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发表时间：
2022-06-14
影响因子：
7.4
通讯作者：
Bleiholder, Christian
中科院分区：
文献类型：
--
作者：
Liu, Fanny C.;Kirk, Samuel R.;Caldwell, Kirsten A.;Pedrete, Thais;Meier, Florian;Bleiholder, Christian
自上而下的蛋白质组学提供了一种直接的方法，蛋白质的水平，但仍然是技术上的挑战。使用离子迁移谱/质谱（IMS/MS）分离自上而下的碎片离子改善了信号/噪声和动态范围。然而，这样的应用程序，还没有充分利用从IMS/MS，这是通过测量的动量转移截面的碎片离子结构的表征获得的主要信息。在这里，我们使用串联捕获离子迁移谱仪/质谱仪（tTIMS/MS）对完整的蛋白质和组件进行自上而下的分析，并编译超过1400个碎片离子的横截面值。我们的分析表明，大多数碎片离子表现出多种，稳定的构象类似于完整的多肽和蛋白质。数据进一步表明，构象异质性强烈影响的片段离子的氨基酸序列。此外，时间分辨tTIMS/MS实验表明，自上而下的碎片离子的构象可以是亚稳态的离子迁移率测量的时间尺度上。两者合计，我们的分析表明，自上而下的碎片离子在气相中进行折叠过程，这种折叠过程可以导致动力学捕获的中间态离子迁移率测量。因此，由于多肽离子的折叠自由能表面是由其氨基酸序列和电荷状态编码的，我们的分析表明，横截面可以被利用为自上而下的碎片离子的序列特异性决定因素。
Top-down proteomics provides a straightforward approach to the level of proteoforms but remains technologically challenging. Using ion mobility spectrometry/mass spectrometry (IMS/MS) to separate top-down fragment ions improves signal/noise and dynamic range. Such applications, however, do not yet leverage the primary information obtained from IMS/MS, which is the characterization of the fragment ion structure by the measured momentum transfer cross sections. Here, we perform top-down analysis of intact proteins and assemblies using our tandem-trapped ion mobility spectrometer / mass spectrometer (tTIMS/MS) and compile over 1400 cross section values of fragment ions. Our analysis reveals that most fragment ions exhibit multiple, stable conformations similar to those of intact polypeptides and proteins. The data further indicate that the conformational heterogeneity is strongly influenced by the amino acid sequences of the fragment ions. Moreover, time-resolved tTIMS/MS experiments reveal that conformations of top-down fragment ions can be metastable on the timescale of ion mobility measurements. Taken together, our analysis indicates that top-down fragment ions undergo a folding process in the gas-phase and that this folding process can lead to kinetic trapping of intermediate states in ion mobility measurements. Hence, because the folding free energy surface of a polypeptide ion is encoded by its amino acid sequence and charge state, our analysis suggests that cross sections can be exploited as sequence-specific determinants of top-down fragment ions.
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影响因子：
7.4
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通讯作者：
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