Structural Investigation of ¹³C Labeled N-linked Glycans from Trichomonas vaginalis by Electron Transfer Dissociation

Trichomonas vaginalis, which is sexually transmitted, causes vaginitis in women and urethritis in men and increases the risk of HIV transmission. Trichomonas makes a truncated Asn-linked glycan (N-glycan) composed of seven sugars (Man₅GlcNAc₂), which is used for quality control of protein folding in the lumen of the ER. For the samples studied here, metabolic labeling with ¹³C₁-mannose served to efficiently distinguish Trichomonas N-glycans from contaminating N-glycans originating from the fetal bovine serum used to culture the parasites.

The classic fragmentation technique for structural identification of glycans is collision-induced dissociation (CID). However, CID generates product ions primarily by the rupture of glycosidic bonds, and thus provides information only on monomer residues and their sequences. The peaks from cross-ring fragmentation often have very low abundances in such spectra. We are applying a newly developed mass spectrometry fragmentation technique—–electron transfer dissociation (ETD) to structural determinations of glycans.

The N-linked glycans labeled with ¹³C at the C₁ of the Man residues were released from the Trichomonas samples with PNGase F and were permethylated. The magnesium-adducted permethylated glycans were analyzed on a Bruker AmaZon quadrupole ion trap. ETD generated both glycosidic bond cleavage and cross-ring cleavage ions. The cross-ring cleavages clarified the different linkage types and branching patterns of the N-linked glycans. The utilization of ETD-CID MS3 techniques and the presence of the ¹³C₁-labels in the mannose residues made it possible to verify peak assignments and to distinguish the overlapping peaks. These results predict that ETD should contribute to confident structural analysis of a wide variety of oligosaccharides.

This research is supported by NIH grant P41 RR10888. The authors are grateful to Bruker Daltonics for loan of the AmaZon instrument and to our collaborators A. Carpentieri, P. W. Robbins and J. Samuelson for generation of the ¹³C-labeled Trichomonas glycans.