Online Deuterium Hydrogen Exchange and Protein Digestion Coupled with Ion Mobility Spectrometry and Tandem Mass Spectrometry

Online deuteriam hydrogen exchange (DHX) and pepsin digestion (PD) is demonstrated using drift tube ion mobility spectrometry (DTIMS) coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron transfer dissociation.(ETD) capabilities. DHX of deuterated ubiquitin, followed by subsequent quenching and digestion, is performed within similar to 60 s, yielding 100% peptide sequence coverage. The high reproducibility of the IMS separation allows spectral feature matching between two-dimensional IMS-MS datasets (undeuterated and deuterated) without the need for dataset alignment. Extracted ion drift time distributions (XIDTDs) of deuterated peptic peptides are mobility-matched to corresponding XIDTDs of undeuterated peptic peptides that were identified using collision-induced dissociation (CID). Matching XIDTDs allows a straightforward identification and deuterium retention evaluation for labeled peptides. Aside from the mobility separation, the ion trapping capabilities of the LTQ, combined with ETD, are demonstrated to provide single-residue resolution. Deuterium retention for the c- series ions across residues M-1-L-15 and N-25-R-42 in good agreement with the known secondary structural elements within ubiquitin.