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J Biomol Tech. 2007 February; 18(1): 14.
PMCID: PMC2291948

P38-M Improved Characterization of Glycopeptides Using CID and ETD in a Non-Linear Ion Trap MS

Abstract

Characterization of glycoproteins by electrospray ionization tandem mass spectrometry of glycosylated peptides using collisionally induced dissociation (CID) results in the preferential fragmentation of the glycosidic bonds, while the peptide bonds are more stable. As low-energy CID of glycopeptides mainly provides information on the glycan moiety, a selective analysis of the peptide chain itself usually requires enzymatic removal of the carbohydrate part. The recently introduced electron transfer dissociation (ETD) option for ion-trap mass spectrometers shows preferred fragmentation of the peptide backbone, leaving posttranslational modifications widely intact. Thus, a combination of ETD and CID is a highly promising tool for the analysis of glycopeptides.

A high-capacity three-dimensional quadrupole ion-trap MS with ETD option enables the application of the two complementary fragmentation techniques. For fragmentation via ETD, radical anions of fluoranthene are generated in a negative chemical ionization source and added to previously isolated multiply charged peptide cations inside the ion trap. Different glycosylated proteins were analyzed either offline or with on-line nano-LC-MS/MS in the positive-ion mode. Enriched glycopeptides from a tryptic digest of horseradish peroxidase were analyzed offline using nanospray. For an on-line nano LC approach, digests of human IgG3 were chosen.

With ETD and CID, widely complementary data from these glycopeptides were obtained: CID MS/MS spectra showed mainly sugar fragmentation, while ETD MS/MS allowed for selective fragmentation of the peptide backbones with retention of the intact glycan moiety, thereby allowing the facile deduction of peptide sequence and glycan attachment site. In conclusion, glycopeptides were characterized in detail with regard to carbohydrate composition and structure as well as peptide sequence and modification sites. Importantly, this approach can be incorporated into a conventional glycoproteomics work flow, as sensitivity and speed of data acquisition allow the combined use of ETD and CID with direct LC coupling.


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