Fully realizing the capabilities of tandem mass spectrometry (MS/MS) for analysis of glycosylated peptides will require further understanding of the unimolecular dissociation chemistry that dictates their fragmentation pathways. In this context, the overall composition of a given glycopeptide ion is a key characteristic; however, the extent to which the carbohydrate moiety influences the preferred dissociation channels has received relatively little study. Here, the effect of glycan composition on energy-resolved collision-induced dissociation (CID) behavior was studied for a select menu of 30 protonated high mannose type N-linked glycopeptide ions. Groups of analytes which shared a common charge state, polypeptide sequence, and glycosylation site exhibited 50% precursor ion survival energies that varied only slightly as the size and composition of the oligosaccharide was varied. This was found to be true regardless of whether the precursor ion survival energies were normalized for the number of available vibrational degrees of freedom. The practical consequence of this was that a given collision energy brought about highly similar levels of precursor ion depletion and structural information despite systematic variation of the glycan identity. This lack of sensitivity to oligosaccharide composition stands in contrast to other physicochemical properties of glycopeptide ions (e.g., polypeptide composition, charge state, charge carrier) which sharply influence their energy-resolved CID characteristics. On the whole, these findings imply that the deliberate selection of CID energies to bring about a desired range of fragmentation pathways does not necessarily hinge on the nature of the glycan.

Abstract. Fragmentation of glycopeptides in tandem mass spectrometry (MS/MS) plays a pivotal role in site-specific protein glycosylation profiling by allowing specific oligosaccharide compositions and connectivities to be associated with specific loci on the corresponding protein. Although MS/MS analysis of glycopeptides has been successfully performed using a number of distinct ion dissociation methods, relatively little is known regarding the fragmentation characteristics of glycopeptide ions with various charge carriers. In this study, energy-resolved vibrational activation/ dissociation was examined via collision-induced dissociation for a group of related high mannose tryptic glycopeptides as their doubly protonated, doubly sodiated, and hybrid protonated sodium adduct ions. The doubly protonated glycopeptide ions with various compositions were found to undergo fragmentation over a relatively low but wide range of collision energies compared with the doubly sodiated and hybrid charged ions, and were found to yield both glycan and peptide fragmentation depending on the applied collision energy. By contrast, the various doubly sodiated glycopeptides were found to dissociate over a significantly higher but narrow range of collision energies, and exhibited only glycan cleavages. Interestingly, the hybrid protonated sodium adduct ions were consistently the most stable of the precursor ions studied, and provided fragmentation information spanning both the glycan and the peptide moieties. Taken together, these findings illustrate the influence of charge carrier over the energyresolved vibrational activation/dissociation characteristics of glycopeptides, and serve to suggest potential strategies that exploit the analytically useful features uniquely afforded by specific charge carriers or combinations thereof.

Tandem mass spectrometry (MS/MS) is now well-known as a powerful tool for characterizing the primary structures of peptides and proteins; however, in many cases the use of but a single dissociation method provides only a partial view of the amino acid sequences and post-translational modification patterns of polypeptides. While the application of multiple fragmentation methods can be more informative, this introduces the burden of acquiring multiple MS/MS spectra per analyte, thus reducing the effective duty cycle of such methods. In this work, initial proof-of-concept is provided for a method designed to overcome these barriers. This method relies on the complementary fragmentation information that can be provided by performing collision-induced dissociation (CID) and electron transfer dissociation (ETD) in concert, while also taking advantage of an ion mobility (IM) dimension to temporally resolve the occurrence of CID and ETD when applied to a single accumulated packet of precursor ions. In this way, the significant proportion of the precursor ion population that remains unreacted in ETD experiments is subjected to CID rather than being fruitlessly discarded. In addition, the two distinct fragmentation spectra can be extracted from their corresponding IM domains to render readily interpretable individual fragmentation spectra. This scheme was demonstrated for several polypeptides ranging from 1.3 to 8.6 kDa in molecular weight. In each case, IM-resolved CID and ETD events resulted in b/y and c/z ions, respectively, which each covered both unique and overlapping sequence information. These findings demonstrate that the combination of CID and ETD can be achieved with greater utilization of the available ion population and little or no loss of duty cycle.