on 06-13-201801:44 AM - edited on 10-15-202109:06 AM by AnalyteGuru
Terry Zhang, Stephane Houel, Jonathan Josephs
Purpose: Study protein conformation by hydrogen/deuterium exchange mass spectrometry (HDX) using electron transfer dissociation (ETD) to get single amino acid resolution.
Methods: A model peptide was used to confirm the proper ETD experimental condition for low deuterium scrambling and back exchange. Apo- and holo forms of calmodulin were studied by topdown and bottom-up HDX-ETD experiments using a fully automatic HDX workflow station with a Thermo Scientific™ Orbitrap Fusion™ Lumos™ mass spectrometer. A full scan (MS) and a ETD MS2 scan were both conducted at the same time for bottom-up experiments. The MS full spectra were used to probe region of significant change in deuterium incorporation, and the ETD MS2 spectra were then used to pinpoint deuterium incorporation at the single amino acid level for the specific peptides that showed significant deuterium change.
Results: 100% sequence coverage was obtained for both top-down and bottom-up ETD MS2 experiments for apo and holo calmodulin. For the top-down experiment, 43 fragments were obtained for apo and holo calmodulin. Overall, the apo calmodulin showed more deuterium incorporation than the holo calmodulin. Nearly single amino acid deuterium incorporation results were obtained from the consecutive N and C terminal fragments from the top-down experiment. Similar deuterium incorporation pattern were obtained between the top-down and the bottom-up experiment.