Can ETD accurately measure the H/D exchange of individual residues in a protein? - a 'HX HX-MS/MSMS/MS' study of the amyloidogenicamyloidogenic protein beta-2-microglobulin

摘要

By combining automated ETD-MS/MS experiments with a classical HX-MS setup we have established an HX-MS/MS method capable of measuring deuterium levels of both peptide segments and individual residues, thus greatly enhancing the utility of mass spectrometry to monitor the H/D exchange of proteins. Employing the described method, we have monitored the H/D exchange of the amyloidogenic protein beta-2-microglobulin, resolving incorporated deuterium to 44 individual residues located in 18 peptides that cover 96percent of the sequence of beta-2-microglobulin. Deuterium levels of single residues derived from ETD fragment ions of doubly and triply charged precursors were found to accurately correlate with hydrogen exchange rates measured by NMR spectroscopy. The NH_(3)-loss of the precursor ion in ETD spectra of deuterium labeled peptides constitute a highly useful internal probe of the occurence of scrambling in peptide ions prior to ETD during HX-MS/MS experiments.