Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections

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 l...

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Published in:	Analytical chemistry (Washington) Vol. 94; no. 23; pp. 8146 - 8155
Main Authors:	Liu, Fanny C., Kirk, Samuel R., Caldwell, Kirsten A., Pedrete, Thais, Meier, Florian, Bleiholder, Christian
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 14-06-2022
Subjects:	Amino acid sequence Amino acids Chemistry Cross-sections Folding Free energy Heterogeneity Ion Mobility Spectrometry - methods Ionic mobility Ions Ions - chemistry Mass spectrometry Mass spectroscopy Mobility Momentum transfer Nucleotide sequence Peptides Polypeptides Proteins Proteins - chemistry Proteomics Proteomics - methods Scientific imaging Spectroscopy Tandem Mass Spectrometry - methods Vapor phases
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 current address: Incyte Research Institute, Department of Chemistry, 1801 Augustine Cut-off, Wilmington, DE 19803 Author Contributions current address: School of Information, Florida State University, 142 Collegiate Loop, Tallahassee, FL 32306 F.C.L, C.B. conceived the study. F.C.L., S.R.K., K.A.C, T.P. performed experiments. C.B. developed the data processing algorithm and C.B., F.C.L., K.A.C., S.R.K. analyzed the measurements and compiled the cross-sections. C.B., F.C.L. performed the computational modelling. C.B., F.C.L., S.R.K. prepared the initial draft and figures. F. M. and C.B. discussed the compiled data. C.B., F.M., and F.C.L. co-wrote the manuscript.
ISSN:	0003-2700 1520-6882
DOI:	10.1021/acs.analchem.1c05171