Precursor ion survival energies of protonated N-glycopeptides and their weak dependencies on high mannose N-glycan composition in collision-induced dissociation

Abstract

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.

ExpandingN-Glycopeptide Identifications by Fragmentation Prediction and Glycome Network Smoothing

“…This also highlights the importance of the proton mobility feature. In low proton mobility settings, larger peptide+Y ions tended to be much more abundant, and all peptide+Y ions were present at higher charge states compared to high proton mobility cases, consistent with previous work [34, 6]. Our model seemed to perform well on the high mannose N -glycans of the mouse brain tissue dataset, but performed less consistently on sialylated glycans.…”

“…The mobile proton hypothesis is a widely accepted kinetic model of fragmentation for protonated peptides [33, 35] which has been used to create many peptide fragmentation prediction algorithms [21, 32]. Unsurprisingly, glycopeptide fragmentation depends on mobile protons as well, driving very different abundance patterns of fragmentation depending upon charge state [34, 6]. We used the proton mobility classification scheme described in [32], where the number of K, R , and H are compared to the precursor ion’s charge, where if the sum is greater than the charge, the precursor is immobile , if equal, the precursor is partially mobile , or less than, the precursor is mobile.…”

“…Mass spectrometry has been established as one of the best tools for high throughput analysis of the glycoproteome [4]. Intact glycopeptide tandem mass spectrometry (MS/MS) interpretation is a challenging problem to address as data is generated with a variety of dissociation strategies and energies, and depending upon their appropriateness for different types of glycopeptides in glycoproteomes large and small [5, 6, 7, 8].…”

“…Collisionally dissociated glycopeptide MS/MS spectra are complex, having peptide b and y ions with or without glycan reducing end residues, glycan B ions, and intact peptide + glycan Y ions, with varying abundances in varying charge states depending upon the number and strength of the bonds in the precursor molecule and the collision energy used [6]. Thus far only glycan B ions have received critical analysis about their inter-relationships [14, 15] and limited work has been done on broad glycopeptide spectrum prediction [16] while these topics have been well explored and exploited in peptide spectrum matching [17, 18, 19, 20, 21, 22, 23].…”

Parallel Determination of Polypeptide and Oligosaccharide Connectivities by Energy-Resolved Collison-Induced Dissociation of Protonated O-Glycopeptides Derived from Nonspecific Proteolysis

“…First, the collision energies at which the precursor ion is reduced to 50% relative intensity are significantly lower for these doubly protonated Oglycopeptides than for doubly protonated N-glycopeptides of similar molecular weight. For example, a previous ER-CID study of doubly protonated N-glycopeptides ranging from m/z 967.9 to 1346.9 demonstrated 50% precursor ion survival at ΔU values of approximately 27.5−47.5 V. 52 By contrast, in the present study the doubly protonated O-glycopeptides ranged from m/z 869.2 to 1131.8 and exhibited 50% precursor ion survival energies at at ΔU values of about 8.0−11.0 V. This also suggests the possibility that the tuning of collision energies to achieve a desired level of fragmentation may be more sensitive for O-glycopeptides than for N-glycopeptides. Second, the ΔU settings resulting in approximately 50% precursor ion survival are within just a few volts of one another for these three exemplars despite the >500 u difference in molecular weight (and attendant difference in available degrees of vibrational freedom) among them.…”