Abstract
Mass spectrometric analysis of intact glycopeptides can reveal detailed information about glycosite, glycan structural features, and their heterogeneity. Sialyl glycopeptides can be positively, negatively, or neutrally charged depending on pH of their buffer solution and ionization conditions. To detect sialoglycopeptides, a negative-ion mode mass spectrometry may be applied with a minimal loss of sialic acids, although the positively charged or neutral glycopeptides may be excluded. Alternatively, the sialyl glycopeptides can be identified using positive-ion mode analysis by doping a high concentration of sodium salts to the analytes. Although manipulation of unmodified sialoglycopeptides can be useful for analysis of samples, less than optimal ionization, facile loss of sialyl and unfavorable ionization of accompanying non-sialyl peptides make such strategies suboptimal. Currently available chemical derivatization methods, while stabilizing for sialic acid, mask sialic acid linkage configuration. Here, we report the development of a novel approach to neutralize sialic acids via sequentially chemical modification that also reveals their linkage configuration, often an important determinant in biological function. This method utilizes several components to facilitate glycopeptide identification. These include the following: solid phase derivatization, enhanced ionization of sialoglycopeptides, differentiation of sialic acid linkage, and enrichment of the modified glycopeptides by hydrophilic interaction liquid chromatography. This technology can be used as a tool for quantitative analysis of protein sialylation in diseases with determination of sialic acid linkage configuration.Graphical ᅟ
Highlights
Glycosylation is one of the most abundant posttranslational modifications and is often found on secreted, S
hydrophilic liquid interaction chromatography (HILIC) enrichment without prior ethanol-ethylene diamine (EDA) derivatization resulted in 32% of ion intensity present in identified ions and 0.095% of intensity in the flowthrough; HILIC enrichment with prior ethanol-EDA modification resulted in 58% of ion intensity present in identified ions and 0.037% of intensity in the flow-through. These results demonstrate that ethanol-EDA derivatized glycopeptides can enriched by HILIC chromatography and generate better coverage probably due to the better ionization, interaction with HILIC matrix, as well as stabilization of sialic acids
The α2,3 linkages are derivatized by EDA amidation
Summary
Glycosylation is one of the most abundant posttranslational modifications and is often found on secreted, S. Yang et al.: Derivatization for Sialic Acid Linkage in Glycopeptides methods that can reveal important features of these chemical entities is desirable. As a terminal substitution on many glycans, sialic acid plays a critical role in a range of recognition events in biology. Avian influenza hemagglutinin (HA) primarily recognizes sialylα residues while human influenza favors sialylα substitutions and swine influenza recognizes both [6, 7]. Pandemics can arise when a mutation in avian or swine influenza HA shifts sialyl specificity toward sialylα. A method, such as the one presented here, that can detect these subtle differences, α2,3 versus α2,6-linked sialic acids, at the glycosylation site of the resident protein can provide an important tool to better understand the role of sialic acids in such biological processes
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