On‐target endoglycosidase digestion matrix‐assisted laser desorption/ionization mass spectrometry of glycopeptides

Abstract

The digestion of glycopeptides with endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 degrees C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The endoglycosidases used for digestion were endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger.

Matrix‐assisted laser desorption/ionization on‐target method for the investigation of oligosaccharides and glycosylation sites in glycopeptides and glycoproteins

“…However, it has been shown that the cleavage of monosaccharide moieties from oligosaccharides on the target can be done by some exoglycosidases in the presence of 2‐aza‐2‐thiothymine 21. In another study, digestion with endoglycosidases was performed in the presence of sinapinic acid, a matrix commonly used for the analysis of glycopeptides and MALDI mass spectra provided ions with m/z values corresponding to the deglycosylated peptides 22…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update covering the period 2001–2002

“…Studies on the rate of release of several N-linked glycans with PNGase F or endo-H on the MALDI target at room temperature have shown that quantitative release from three low-mass glycoproteins (up to 5.7 kDa) can take as little as 10 min both with and without the presence of the sinapinic acid (1/48) matrix. However, the released glycans were not studied in this report (Colangelo & Orlando, 2001). Membrane-bound viral glycoproteins, solubilized with n-octyl-glucoside (10), have been deglycosylated with PNGase F at 238C on the MALDI target coated with acidresistant hydrophobic tape to avoid detergent-induced spreading, and both the protein and glycoprotein examined from sinapinic acid to define glycosylation.…”

Site-specific N-glycan characterization of human complement factor H

“…• C. On-target deglycosylation of the glycopeptides was performed (Colangelo and Orlando 2001). Briefly, 0.5 µL of the Glycosylation analysis of human complement factor H LC fraction dried and resuspended in water, 0.5 µL of PNGase F solution, and 1.5 µL of water were thoroughly mixed on the MALDI target.…”