Matrix‐assisted laser desorption/ionization mass spectrometry compatible β‐elimination of O‐linked oligosaccharides

Abstract

A new beta-elimination procedure has been introduced to cleave O-linked oligosaccharides from low- to sub-microgram amounts of glycoproteins prior to analysis by mass spectrometry. Borane-ammonia complex in aqueous ammonia is used as a cleaving solution alternative to the sodium borohydride/sodium hydroxide medium conventionally used in beta-elimination. The procedure results in minimum sample purification, leading to minimal sample loss and consequently an overall enhancement in sensitivity. It was applied successfully in the analysis of bovine fetuin and submaxillary mucin, as well as to a complex bile-salt-stimulated lipase glycoprotein isolated from human milk.

Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins

“…We have shown previously that the reaction of an aqueous solution of ammonia-borane complex with carbohydrates quantitatively reduces mono-and oligosaccharides irrespective of their nature [51].…”

“…However, this aspect of the separation is not desired in the quantitative analysis of mixtures of monosaccharides, since each free-reducing end monosaccharide appears as two peaks (␣-and ␤-anomers). Free reducing end monosaccharides and glycans are easily reduced using ammoniaborane complex reagent [51].…”

“…The samples were then vacuum dried, dissolved in 50 L water, and dried again. Reduction to the corresponding alditols was performed according to our previously published procedure [51]. Briefly, a 10-L aliquot of a 10 mg/mL aqueous ammonia-borane complex solution was added to each sample, followed by incubation at 60°C in a water bath for 1 h. A 10-L aliquot of 5% acetic acid was added to destroy any remaining ammonia-borane complex, and the samples were vacuum dried.…”

Analysis of protein glycosylation by mass spectrometry

“…To prevent the denaturation of PNGase F, a non-ionic detergent (nonidet P40, NP40) is added before digestion with the deglycosylation enzyme. After PNGase F digestion, the N-deglycosylated protein is precipitated and the N-glycans are purified using a Sep-Pak C 18 cartridge to remove the detergents, which may considerably affect the quality of the MS spectra. With less than 50 mg of protein, sequential MALDI profiling, MS-MS sequencing, linkage analysis and exoglycosidase digestions on the MALDI target can normally be performed.…”

“…This method has worked well in our hands. Other methods, including gel reductive b-elimination, have been described [16][17][18][19] .…”