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submitted on 06.04.2018 and posted on 06.04.2018by 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.