Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography

Urs Lewandrowski,Katharina Lohrig,Dirk Wolters,René P Zahedi,Albert Sickmann

doi:10.1007/s12014-008-9006-z

Abstract

Abstract Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.

Highlights

Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated
Glycopeptides have been demonstrated to exhibit a similar behavior as phosphopeptides in other systems such as strong cation exchange [13, 22] and titanium dioxide chromatography [15, 23]
Carbohydrates are responsible for enabling, e.g., cell–cell recognition and cellular adhesion caused by their presence on membrane glycoproteins

Summary

Introduction

Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Being a very heterogeneous group of modifications, a narrow focus of studies on certain subsets of glycosylation types is required In this context, the majority of glycosylation site analyses were focused on Nglycosylations in the past, demonstrating the strong impact of glycosylations on protein and cellular function. The majority of glycosylation site analyses were focused on Nglycosylations in the past, demonstrating the strong impact of glycosylations on protein and cellular function They influence protein structure as well as their subcellular localization, e.g., by controlling correct folding of newly synthesized proteins and transport signals like mannose-6-. The impaired synthesis of oligosaccharide side chains of membrane glycoproteins has been shown to be connected to congenital thrombocytopenia [4] as shown for the terminal glycosylation of glycoprotein Ib in platelets [5, 6]

Methods

Results

Conclusion