Glycoprotein methods protocols - biotechnology 048-9-121-128.pdf

Sequencing Glycopeptides 121

121

From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A. Corfield © Humana Press Inc., Totowa, NJ

11

Identification of Glycosylation Sites in Mucin Peptides

by Edman Degradation

Natasha E. Zachara and Andrew A. Gooley

1. Introduction

Although it is possible to determine and characterize the total carbohydrate profile

after release of the mucin oligosaccharides (usually by β-elimination), it is challeng￾ing to assign the sites of glycosylation (macroheterogeneity) and the carbohydrate

heterogeneity at a given glycosylation site (microheterogeneity). Typically, the char￾acterization of macro- and microheterogeneity has been dependent on the isolation of

small peptides with only one glycosylation site. However, this is not possible with

high molecular weight, heavily glycosylated domains such as those found in mucins.

The two methods for determining the sites of glycosylation in proteins are mass

spectrometry and Edman sequencing. Multiple sites of glycosylation cannot easily be

detected using mass spectrometry; one strategy involves the β-elimination of the car￾bohydrate, which results in the conversion of serine to dehydro-alanine and threonine

to α-amino butyric acid. These amino acids have unique masses and can be used to

map glycosylation sites (1,2). However, the macro- and microheterogeneity of the

carbohydrates can not be determined unless the peptide has just one glycosylation site.

In 1950, Edman sequencing was introduced as a repetitive degradation of proteins

with phenylisothiocyanate (3). In the mid-1960s, the process was automated (4),

resulting in a machine where the N-terminal amino acid is derivatized, cleaved, and

transferred to a separate reaction vessel, in which it undergoes a conversion to a

phenylthiohydantoin (PTH)-amino acid (Fig. 1). It is the PTH-amino that is separated

by reversed-phase chromatography and detected.

Although the modern pulsed liquid sequenator is pmol sensitive, glycosylated

amino acids are only recovered in low yield. Samples are sequenced (Fig. 1) on glass￾fiber supports or membranes such as polyvinylidene difluoride (PVDF). Following

cleavage, the released amino acid is transferred from the reaction cartridge to the con￾version flask by nonpolar solvents such as ethyl acetate or chlorobutane. The more