Characterization of glycopeptides by combining collision‐induced dissociation and electron‐transfer dissociation mass spectrometry data

Abstract

Structural characterization of a glycopeptide is not easily attained through collision-induced dissociation (CID), due to the extensive fragmentation of glycan moieties and minimal fragmentation of peptide backbones. In this study, we have exploited the potential of electron-transfer dissociation (ETD) as a complementary approach for peptide fragmentation. Model glycoproteins, including ribonuclease B, fetuin, horseradish peroxidase, and haptoglobin, were used here. In ETD, radical anions transfer an electron to the peptide backbone and induce cleavage of the N-Calpha bond. The glycan moiety is retained on the peptide backbone, being largely unaffected by the ETD process. Accordingly, ETD allows not only the identification of the amino acid sequence of a glycopeptide, but also the unambiguous assignment of its glycosylation site. When data acquired from both fragmentation techniques are combined, it is possible to characterize comprehensively the entire glycopeptide. This is being achieved with a mass spectrometer capable of alternating between CID and ETD on-the-fly during an LC/MS/MS analysis. This is demonstrated here with several tryptic glycopeptides.

A Comparison of Energy-Resolved Vibrational Activation/Dissociation Characteristics of Protonated and Sodiated High Mannose N-Glycopeptides

“…Substantial effort has been directed towards exploiting the specific advantages afforded by different ion dissociation methods for glycopeptide analysis, including those based on ion-neutral interactions, as in collision-induced dissociation (CID) [14][15][16][17][18]; ion-electron and ion-ion reactions, as in electron capture dissociation (ECD) and electron transfer dissociation (ETD) [19][20][21][22]; and irradiation with photons, as in infrared multiphoton dissociation (IRMPD) and ultraviolet photodissociation (UVPD) [23][24][25][26]. By comparison, less attention has been paid to the effects of charge carrier upon glycopeptide dissociation patterns and energetics.…”

Improved Peptide Identification for Proteomic Analysis Based on Comprehensive Characterization of Electron Transfer Dissociation Spectra

“…17,18 On these instruments, it has been demonstrated that ETD is complementary to CID and is favored over CID to analyze large peptides, proteins, and labile post-translational modifications (PTMs). [15][16][17][18][19][20][21][22][23] The past few years have seen a rapid increase of proteomic applications employing ETD, such as analysis of phosphorylation, 24,25 glycosylation, 26,27 quantification, 28,29 or top-down proteomics. 30,31 In contrast to the rapid advancement of instrumentation and application, the informatics research on ETD has lagged behind, impeding its full potential.…”

Glycan Analysis by Ion Mobility–Mass Spectrometry

“…Whereas collision-induced dissociation (CID) predominantly produces glycan fragments,e lectrontransfer dissociation (ETD) can be applied to fragment the peptide backbone. [33] Similar to the analysis of intact glycans,i tw as demonstrated that intact glycopeptide isomers can be differentiated by using IM-MS. [32,34] More importantly,h owever,t he previously described fragment approach can also be used. Recently,g lycopeptides differing merely in the regiochemistry of as ingle glycosidic bond within the glycan were investigated.…”