Strategy for Profiling and Structure Elucidation of Mucin-Type Oligosaccharides by Mass Spectrometry

Abstract

A strategy combining accurate mass determination, tandem mass spectrometry, structure homology, and exoglycosidases is described that allows the structural characterization of mucin-type O-linked oligosaccharides. The method is used to profile with quantitation the O-linked oligosaccharide (both neutral and anionic) components of the only diploid Xenopus frog, Xenopus tropicalis. Collision-induced dissociation was used to determine connectivity, to identify previously characterized oligosaccharides, and to determine the presence of structural motifs in unknown oligosaccharides. Exoglycosidase digestion was used to identify the individual residues along with the linkages. The enzymes were also used to cleave larger oligosaccharides to smaller units that are similar to previously elucidated components. By using CID, isomeric structures were compared to determine whether they were identical. In this way, the exoglycosidases were more effectively used, and their use was minimized. A total of 35 oligosaccharides including neutral, sialylated, and sulfated were characterized in this way. The relative abundances of all components were also determined based on HPLC.

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

“…An aliquot of 50 μg purified diSA-proteins (or unmodified proteins resuspended in PBS) was either treated with PNGase F to release the N-glycans or subjected to β-elimination to release O-linked glycans (41,42). The resulting N-and O-linked oligosaccharides were loaded on PGCs and then washed with water (3.0 mL) to remove buffer and salts.…”

Mass spectrometric profiling of N-linked oligosaccharides and uncommon glycoform in mouse serum with head and neck tumor

“…Moreover, carboxylic groups cause sialylated oligosaccharides to ionize with less efficiency than neutral oligosaccharides, making MS analysis of glycans more difficult in the positive ion mode [16]. Neutral oligosaccharides can be separated from acidic ones by various techniques before analysis [17,18], or to chemically modify the carboxyl group by methods such as methyl esterification [19], permethylation [20], or amidation [21].…”

New hyphenated methodologies in high‐sensitivity glycoprotein analysis

“…As the low-energy CID in the IT analyzers [32,34,44] or the PSD in MALDI/TOF MS instruments [45] is generally insufficient to accomplish this fragmentation task, we have chosen a MALDI/TOF/TOF MS with high-energy CID capabilities to furnish extensive fragmentation of model neutral [46][47][48][49][50] and acidic [46,51] glycans. An alternative approach pursued elsewhere for extensive fragmentation of glycans involves Fourier transform ion cyclotron resonance MS used in conjunction with sustained offresonance irradiation (SORI) [52], CID [52,53], IR multiphoton dissociation (IRMPD) [54][55][56], or electron capture dissociation (ECD) [54,55]. In our MALDI/TOF/TOF MS experiments, we were able to distinguish a subtle form of isomerism in the mannose-rich glycans [47] (Fig.…”

“…Moreover, these columns are unaffected by strongly acidic or alkaline conditions and can be used throughout the entire pH range with a wide range of solvents. Thus far, these columns have been utilized in conjunction with MS for the analysis of glycans derived from different samples, including egg jelly surrounding Xenopus laevis eggs [53,75,76], human bronchial epithelial cell cultures [77], membrane proteins from premature aging HuchinsonGilford progeria syndrome fibroblasts [78], human tear fluid [79], and human plasma [80]. Moreover, microliter flow rates were employed in all these applications, allowing only the detection of structures present in abundance.…”