Analysis of Carbohydrates on Proteins by Offline Normal-Phase Liquid Chromatography MALDI-TOF/TOF-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 136

Chapter 8

Analysis of Carbohydrates on Proteins by Offline
Normal-Phase Liquid Chromatography
MALDI-TOF/TOF-MS/MS

Theodora Tryfona and Elaine Stephens

Abstract

Glycoproteins typically exist as a diverse population of glycoforms, which can consist of a great number of
different glycan structures (including structural isomers) that vary in their degree of occupancy at given
glycosylation sites. Hence, the detailed characterization of such glycans can be a very demanding task.
Liquid chromatography in combination with mass spectrometric techniques can provide a fast and sensi-
tive tool for the analysis of these structurally complex molecules. Here we describe a sensitive method that
employs capillary normal-phase HPLC coupled offline to MALDI-TOF/TOF-MS/MS for the detailed
characterization of enzymatically released N -glycans. The normal-phase chromatography allows the sep-
aration of some isobaric glycan structures and analysis of the glycans by MALDI high-energy CID gives
sequence, linkage and branching information.

Key words: Protein glycosylation, N -glycans, MALDI-TOF-MS, MALDI-CID, normal-phase
chromatography.

1. Introduction

Glycan chains on proteins play key roles in several biological
processes ( 1 ) , such as cell adhesion, protein folding, solubility
and aggregation, and therefore are of great biological interest.
Nearly all glycoproteins consist of glycoforms in which a sin-
gle protein is diversified by a heterogeneous array of glycans at
each glycosylation site. Detailed characterization of the glycans
attached to proteins therefore depends on the availability of sen-
sitive methods that are capable of successfully analysing mixtures.
Mass spectrometry is one such method and has been extremely