Title

Protonation isomers of highly charged protein ions can be separated in FAIMS-MS

RIS ID

145674

Publication Details

Zhang, J., Donor, M., Rolland, A., Leeming, M., Wang, H., Trevitt, A., Kabir, K., Prell, J. & Donald, W. (2020). Protonation isomers of highly charged protein ions can be separated in FAIMS-MS. International Journal of Mass Spectrometry, 457

Abstract

© 2020 Elsevier B.V. High-field asymmetric waveform ion mobility spectrometry-mass spectrometry (FAIMS-MS) can resolve over an order of magnitude more conformers for a given protein ion than alternative methods. Such an expansion in separation space results, in part, from protein ions with masses of >29 kDa undergoing dipole alignment in the high electric field of FAIMS, and the resolution of ions that adopt pendular vs free rotor states. In this study, FAIMS-MS, collision-induced dissociation (CID), and travelling wave (TW) IMS-MS were used to investigate the pendular and free rotor states of protonated carbonic anhydrase II (CAII, 29 kDa). The electrospray ionization additive 1,2-butylene carbonate was used to increase protein charge states and ensure extended ion conformations were formed. For relatively high charge states in which dipole alignment occurs (30–38+), FAIMS-MS can baseline resolve the isobaric pendular and free rotor ion populations. For TWIMS-MS, these same charge states resulted in monomodal arrival time distributions with collision cross sections corresponding to highly extended ion conformations. Interestingly, CID of FAIMS-selected pendular and free rotor ion populations resulted in significantly different fragmentation patterns. For example, CID of the dipole aligned CAII 37+ resulted in cleavages C-terminal to residue 183, 192 and 196, whereas cleavage sites for the free rotor population occurred near residues 12 and 238. Given that the cleavage sites are ’directed’ by protonation sites in the CID of protein ions, and highly charged protein ions adopt extended conformations with the same or very similar collision cross sections, these results indicate that the pendular and free rotor populations separated in FAIMS can be attributed to protonation isomers. Moreover, the extent of protein ion charging in FAIMS-MS decreased substantially as the carrier gas flow rate decreased, indicating that ion charging in FAIMS-MS can be limited by proton-transfer reactions. Given that the total mass of proton charge carriers corresponds to less than 0.2% the mass of CAII, we anticipate that FAIMS-MS can be used to separate intact isobaric proteoforms with masses of at least ∼29 kDa that result from alternative sites of post-translational modifications.

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Link to publisher version (DOI)

http://dx.doi.org/10.1016/j.ijms.2020.116425