Use of Ion Mobility and Fragment Ions to Increase Coverage in Hydrogen-Deuterium Exchange

Peptide signals in H/D exchange mass spectrometry experiments are often overlapping on the m/z scale due to the nonspecific digestion, fast chromatographic conditions, and cold temperatures required to preserve the deuterium label. Co-eluting peptides which are well resolved when undeuterated can overlap and become impossible to analyze with increasing time in deuterium. It has been shown that the addition of ion mobility into the LC-MS analysis of deuterium labeled samples enables an orthogonal separation in the gas phase. This added separation power is poised nicely between chromatographic and mass spectral analyses (1). The use of ion mobility in H/D experiments resolves many peptides that show spectral overlap. This increases the number of peptides that can be followed in an H/D exchange experiment as well as increases the overlapping linear sequence coverage. This becomes more important with increasing sample complexity, protein and protein complex molecular weight, and importantly with a desire for an increase in backbone resolution to 1 or 2 amino acids. Previously, fragment ions have been used in data-dependent experiments to measure deuterium uptake (2). In this study, fragment ions that were generated in a data-independent manner (3) were used to measure deuterium uptake. In some cases, precursor ions were obstructed under both MS E and HDMS E conditions. However, fragment ions from these peptides sometimes showed no interference. The use of fragment ions that have had the additional specificity of ion mobility alignment improved the measurement over those that only used mass and retention time to associate precursors with product ions. Comparisons of uptake data for peptides and their fragment ions will be demonstrated.