A novel, ultrasensitive approach for quantitative carbohydrate composition and linkage analysis using nanoLC-ESI ion trap tandem mass spectrometry — ASN Events
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A novel, ultrasensitive approach for quantitative carbohydrate composition and linkage analysis using nanoLC-ESI ion trap tandem mass spectrometry (#101)

Kathirvel Alagesan 1 2 , Daniel Varón Silva 2 , Peter H Seeberger 1 2 , Daniel Kolarich 2
  1. Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
  2. Max Planck Institute for colloids and Interfaces, Berlin, Germany

Abstract

Introduction

Glycosylation is one of the most complex yet common post translational modifications, which drastically enhances the functional diversity of proteins and influences their biological activity. Identification and characterisation of glycans is an important step in correlating glycan structure to its biological function. Structural assignment of glycans (glycomics) and their compositions based on MS analyses is often based on taking well studied glycosylation pathways for granted. Nevertheless, many monosaccharide building blocks are indistinguishable by mass alone and detailed linkage information is also not easily obtained by MS/MS analyses. In particular when studying glycosylation from less well studied organisms such as prokaryotes, monosaccharide analysis is indispensable to profile the composition of complex carbohydrates present in glycoconjugates.

Methods

Carefully designed sequential permethylation and reductive amination steps prior and after acid hydrolysis enable separation and differentiation of the various monosaccharides and their respective linkage positions. Separation of various derivatised monosaccharides was achieved using Reversed Phase – Liquid Chromatography (RP-LC)-ESI-MS/MS. In addition, absolute quantitation is accomplished including a set of internal standards, thus providing qualitative and quantitative information on the monosaccharide residues present in a specific sample.

Results

Various derivatised monosaccharides alditols were identified based upon their retention time along with mass spectrometric detection. The resulting fragment ions observed in the MS/MS spectra arise from the cleavage of methyl groups are diagnostic for the each substitution pattern. These differences allowed establishment of ‘fragmentation pattern’ for these derivatised monosaccharide alitols. Furthermore, we have established fragmentation pattern library for various biologically important N-glycans for automated linkage analysis.

Conclusion

The method reported herein has a limit of detection of ≤ 250 fmol for all the monosaccharide analysed and is sensitive to as low as 12 pmol of initial analyte, which now allows to perform both, glycomics and monosaccharide analyses from
low µg amounts of initial glycoprotein. The present method reported here does provide more confidence in correlating the structure and stereochemistry of monosaccharides present in any glycoconjugates, thereby increasing the confidence in glycan structure assignments obtained from glycomics experiments.

Novel Aspect

The methodology  reported describes for the first time, a simple and sensitive method using Reversed Phase – Liquid Chromatography (RP-LC)-ESI-MS/MS for unambiguous identification and linkage determination of various monosaccharides (including N-acetylneuraminic acids).