Top-down Hydrogen/Deuterium Exchange Using Electron Transfer Dissociation for Resolving Backbone Amide Hydrogen Exchange at the Single Single-Residue Level

摘要

Electron transfer dissociation of the model peptide and cytochrome c provided reproducible, complete sequence coverage for the peptide and 80percent coverage for the protein. Once the methods were validated on unlabeled standards, backbone amide hydrogen exchange rates were analyzed at the residue level using ETD. Ionization source and ion optics of mass spectrometer were optimized to minimize hydrogen scrambling. Comparison of the CID results with those obtained from ETD fragmentation highlight the scrambling of amide protons in the former. ETD results for the model peptide indicate a prevalence of slow-exchanging protons in the C-terminus of the peptide surface. These results support the non-scrambling nature of backbone amide protons during ETD fragmentation. The high resolving power of the LTQ Orbitrap Velos MS allowed the detection of isotopic envelopes for the ETD-generated fragment ions and enabled unambiguous identification as well as mass-shift calculations for the labeled and unlabeled samples. Its application on the intact protein suggests C-terminus of cytochrome c (90-104) as a better-protected region. In contrast, the region between residues 80 and 89 was less protected, showing faster exchange rates with solvent. The exchange data for cytochrome c confirms selective deuterium incorporation as documented by other studies.