Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published August 23, 2015 | Accepted Version + Supplemental Material
Journal Article Open

A cationic cysteine-hydrazide as an enrichment tool for the mass spectrometric characterization of bacterial free oligosaccharides


In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways.

Additional Information

© 2015 Springer-Verlag Heidelberg. Received: 3 February 2015; revised: 7 May 2015; accepted: 21 May 2015. We thank Sarkis Mazmanian (Caltech) for providing support for bacterial culture growth. This work was supported by a Searle Scholar Fellowship and a Burroughs-Wellcome Fund Career Award to WMC. The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through Grant GBMF775 and the Beckman Institute.

Attached Files

Accepted Version - nihms713561.pdf

Supplemental Material - 216_2015_8798_MOESM1_ESM.pdf


Files (1.3 MB)
Name Size Download all
381.4 kB Preview Download
875.2 kB Preview Download

Additional details

August 22, 2023
October 23, 2023