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

Jang, Kyoung-Soon and Nani, Roger R. and Kalli, Anastasia and Levin, Sergiy and Müller, Axel and Hess, Sonja and Reisman, Sarah E. and Clemons, William M., Jr. (2015) A cationic cysteine-hydrazide as an enrichment tool for the mass spectrometric characterization of bacterial free oligosaccharides. Analytical and Bioanalytical Chemistry, 407 (20). pp. 6181-6190. ISSN 1618-2642. PMCID PMC4539134. doi:10.1007/s00216-015-8798-8.

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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.

Item Type:Article
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URLURL TypeDescription CentralArticle ReadCube access
Levin, Sergiy0000-0003-2242-5459
Hess, Sonja0000-0002-5904-9816
Reisman, Sarah E.0000-0001-8244-9300
Clemons, William M., Jr.0000-0002-0021-889X
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.
Funding AgencyGrant Number
Searle Scholars ProgramUNSPECIFIED
Burroughs-Wellcome FundUNSPECIFIED
Gordon and Betty Moore FoundationGBMF775
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:Enrichment; Free oligosaccharide; Campylobacter; Hydrazide; Glycomics
Issue or Number:20
PubMed Central ID:PMC4539134
Record Number:CaltechAUTHORS:20150701-133700441
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Official Citation:Jang, KS., Nani, R.R., Kalli, A. et al. Anal Bioanal Chem (2015) 407: 6181. doi:10.1007/s00216-015-8798-8
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:58741
Deposited On:01 Jul 2015 22:42
Last Modified:01 Jun 2022 23:19

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