Synthesis and evaluation of carboxymethyl chitosan for glycopeptide enrichment

• Synthesis of carboxymethyl chitosan (CMCH) as a pull-down stationary phase.
• Use of CMCH to enrich glycopeptides from a 12 protein mixture for MS analysis.
• Compared to a commercially available glycopeptide enrichment kit.
• Use of tandem mass tags (TMT) for relative quantitative comparison.
• CMCH able to enrich more sialylation than the commercial kit.

Glycans are known to be involved in a variety of biological processes throughout human physiology. Mass spectrometry has demonstrated itself as powerful analytical tool for quantitative and structural characterization of glycans. Studying these molecules at the glycopeptide level however, offers distinct advantages, namely the ability to characterize both the glycan and peptide fragments simultaneously, and moreover the ability to assign site specific heterogeneity. In light of this, peptides often dominate the spectrum and hinder the ionization efficiency of glycopeptides. For this reason, enrichment protocols prior to downstream MS analysis need to be developed. Here, we discuss the synthesis and use of carboxymethyl chitosan (CMCH) to enrich glycopeptides from a 12 protein mixture for MS analysis. This protocol was compared to a commercially available glycopeptide enrichment kit offered by EMD Millipore through the use of tandem mass tags (TMT) for relative quantification. Using this approach, we identified 98 unique N-linked glycopeptides and observed, that CMCH was able to enrich more sialylation than the commercial kit. In addition, we observed a trend based on TMT reporter ratios with respect to increasing sialylation. This corroborated that this stationary phase was exhibiting a mixed-mode enrichment through both hydrophilic interaction liquid chromatography (HILIC) and weak anion exchange (WAX) principles.

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