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Initial protein unfolding events revealed by 213 nm UVPD coupled to IM-MS
preprintsubmitted on 06.04.2018, 10:49 and posted on 06.04.2018, 19:53 by Alina Theisen, Rachelle Black, Davide Corinti, Jeff Brown, Bruno Bellina, Perdita Barran
In this work we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation, experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M+7H]7+, cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions while cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles.