“One-Pot” Methylation in Glycomics Application:  Esterification of Sialic Acids and Permanent Charge Construction

Abstract

A simple and rapid "one-pot" methylation method to esterify sialic acids and construct a permanent charge was developed for N-linked glycan analysis, which combined complete nonspecific proteolytic digestion and methylation. A mixture of Asn-glycans prepared from Pronase E digestion of the glycoprotein was passed through a cation-exchange column to convert carboxylic acids to the Na+ form before being methylated with methyl iodide. Derivatives could be easily purified with a hydrophilic affinity chromatography cartridge. Mass spectrometry analysis was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/TOF. The mass spectrometric data indicated that carboxylic acids were methylated in addition to the formation of a quaternary ammonium in the amino group of asparagine residues. Three model glycoproteins, including ribonuclease B, ovalbumin, and transferrin, were employed to demonstrate the merits of this technique. Results showed that the stabilization of sialic acid was achieved in addition to the formation of a permanent charge. Compared to the analysis of underivatized N-glycans, detection sensitivity improved approximately 10-fold. The new technique was further evaluated with glycan profiling of serum transferrin and proved to be a sensitive method for the characterizing protein glycosylation.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Thus, Liu et al (2007b) have used the reaction of methyl iodide with the sodium salt of carboxylic acids to form methyl esters of sialylated glycans obtained by pronase E digestion of glycoproteins. This proteolysis reaction leaves asparagine attached to the carbohydrate, which, in the presence of the methyl iodide is converted into its trimethyl quaternary ammonium salt.…”

Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications

“…Previously, we reported a controlled methylation strategy for the analysis of Asn-glycans, 18 namely a simple methylation to produce the methyl esters of sialic acids and generate permanent charges on Asn moieties. If a permanent charge can be generated on the C-terminal amino acid of a glycopeptide, its ionization efficiency would improve, perhaps reducing the MS response bias.…”

MALDI mass spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix

“…Previous MALDI-MS studies of gangliosides have demonstrated their propensity for losing their sialic acid residues [26,27]. Therefore, numerous attempts have been made to develop derivatization methods for stabilizing sialic acid, including methylation [16,29,30]. Alternatively, optimized matrix can be helpful in reducing molecular decay of the analytes during laser irradiation.…”