Enhanced Collision Induced Unfolding and Electron Capture Dissociation of Native-like Protein Ions

Varun V. Gadkari, Carolina Rojas Ramírez, Daniel D. Vallejo, Ruwan T. Kurulugama, John C. Fjeldsted, Brandon T. Ruotolo

Research output: Contribution to journalArticlepeer-review

41 Scopus citations


Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (-3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.

Original languageEnglish (US)
Pages (from-to)15489-15496
Number of pages8
JournalAnalytical Chemistry
Issue number23
StatePublished - Dec 1 2020
Externally publishedYes

Bibliographical note

Funding Information:
CIU method development in the Ruotolo group is supported through an Agilent Technologies Applications and Core Technology University Research (ACT-UR) Grant. Additional support for this project was provided by the Agilent Technologies Thought Leader Award and University Relations grant programs. C.R.R. was supported by grant T32 CA140044 from the National Institutes of Health.

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