Liquid Chromatography–Tandem Mass Spectrometry Approach for Determining Glycosidic Linkages

Abstract

The structural analysis of carbohydrates remains challenging mainly due to the lack of rapid analytical methods able to determine and quantitate glycosidic linkages between the diverse monosaccharides found in natural oligosaccharides and polysaccharides. In this research, we present the first LC-MS/MS-based method for the rapid and simultaneous relative quantitation of glycosidic linkages for oligosaccharide and polysaccharide characterization. The method developed employs ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ-MS) analysis performed in multiple reaction monitoring (MRM) mode. A library of 22 glycosidic linkages was built using commercial oligosaccharide standards. Permethylation and hydrolysis conditions along with LC-MS/MS parameters were optimized resulting in a workflow requiring only 50 μg of substrate for the analysis. Samples were homogenized, permethylated, hydrolyzed, and then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) prior to analysis by UHPLC/MRM-MS. Separation by C18 reverse-phase UHPLC along with the simultaneous monitoring of derivatized terminal, linear, bisecting, and trisecting monosaccharide linkages by mass spectrometry is achieved within a 15-minute run-time. Reproducibility, efficacy, and robustness of the method was demonstrated with galactan (Lupin) and polysaccharides within food such as whole carrots. The speed and specificity of the method enables its application toward the rapid glycosidic linkage analysis of oligosaccharides and polysaccharides.

A nonenzymatic method for cleaving polysaccharides to yield oligosaccharides for structural analysis

“…8b ) 54 , 57 . The linkage information was similarly obtained using a separate recently reported workflow for comprehensive linkage analysis 55 , 56 . The monosaccharide analysis employed acid hydrolysis to produce monosaccharides that were subsequently labeled with 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP).…”

“…Linkage analysis was adapted from Galermo et al with the following modifications 55 , 56 . Briefly, fractionated oligosaccharides and a pool of oligosaccharide standards were reacted with saturated NaOH and iodomethane in DMSO.…”

Structures and Metabolic Properties of Bovine Milk Oligosaccharides and Their Potential in the Development of Novel Therapeutics

“…Alternatively, more rapid approaches that do not rely on pre-fractionation use strategic derivatization and subsequent analysis of the hydrolyzed monosaccharides to provide at least partial linkage information. Galermo et al have recently published a method by which the monosaccharides and linkage types present in oligo- and polysaccharides can be determined in a high-throughput manner using a pair of derivatization strategies and LC-MS (21).…”

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides

“…used a long C18 column to investigate the heterogeneity of SARS‐CoV‐2 S protein [86]. Since RP columns exhibit relatively good reproducibility and stability, researchers work on the alkyl columns to separate isomers like standard N‐ glycans, O‐ glycans, oligosaccharides, or less complex structures [79,83,87,88].…”