Fragmentation Characteristics of Neutral N-Linked Glycans Using a MALDI-TOF/TOF Tandem Mass Spectrometer

Electron detachment dissociation (EDD) has recently been shown by Amster and coworkers to constitute a valuable analytical approach for structural characterization of glycosaminoglycans. Here, we extend the application of EDD to neutral and sialylated oligosaccharides. Both branched and linear structures are examined, to determine whether branching has an effect on EDD fragmentation behavior. EDD spectra are compared to collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) spectra of the doubly and singly deprotonated species. Our results demonstrate that EDD of both neutral and sialylated oligosaccharides provides structural information that is complementary to that obtained from both CAD and IRMPD. In all cases, EDD resulted in additional cross-ring cleavages. In most cases, cross-ring fragmentation obtained by EDD is more extensive than that obtained from IRMPD or CAD. Our results also indicate that branching does not affect EDD fragmentation, contrary to what has been observed for electron capture dissociation (ECD). . Unlike most biomolecules, oligosaccharides often exist as several isomeric forms with diverse linkages, and may form linear or branched structures. Complete structural characterization of oligosaccharides requires the determination of constituent monosaccharides, their linkage, sequence, and branching patterns. Given their diversity and structural complexity, structural elucidation of oligosaccharides often relies upon a wide range of analytical methodologies, of which nuclear magnetic resonance spectroscopy and mass spectrometry are two vital techniques. Tandem mass spectrometry (MS/MS) is widely used for glycan structural characterization [5][6][7], due to the advent of instruments that provide high-quality spectra from even low-abundance molecular species.Tandem mass spectra of oligosaccharides consist mainly of glycosidic and cross-ring product ions. Glycosidic cleavage occurs between monosaccharide units and provides information regarding saccharide sequence and branching. Cross-ring cleavages can provide valuable information regarding saccharide linkage, particularly when occurring at branching residues. Several factors are known to affect oligosaccharide fragmentation and the degree of cross-ring fragmentation, such as the ionizing cation, the lifetime of the ion before detection, and the energy deposited into the ion.Typically, neutral oligosaccharides are analyzed in positive-ion mode, by their protonated forms or through metal ion adduction. In addition, chemical derivatization such as permethylation is widely used to increase sensitivity, reduce molecular ion lability, and produce structurally diagnostic product ions [8 -10]. Low-energy activation methods, such as collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD), applied to protonated oligosaccharides results in predominantly glycosidic cleavages. However, oligosaccharides ionized with alkali, alkaline earth, and transition metals often fragment to yield more cro...

Section: Ms/ms Of Neutral Oligosaccharidessupporting

confidence: 80%

Section: Ms/ms Of Sialylated Oligosaccharidesmentioning

confidence: 99%

Electron detachment dissociation of neutral and sialylated oligosaccharides

Adamson

Håkansson

2007

“…Permethylated high mannose N-glycans have been studied by tandem mass spectrometry and their fragmentation patterns are well established [42,43]. RNase B contains exclusively high mannose oligosaccharides.…”

Section: Porous Graphite Lc/ms Of High Mannose N-glycans From Rnase Bmentioning

confidence: 99%

A glycomics platform for the analysis of permethylated oligosaccharide alditols

Costello

Contado-Miller

Cipollo

2007

156

166

This communication reports the development of an LC/MS platform for the analysis of permethylated oligosaccharide alditols that, for the first time, demonstrates routine online oligosaccharide isomer separation of these compounds before introduction into the mass spectrometer. The method leverages a high-resolution liquid chromatography system with the superior fragmentation pattern characteristics of permethylated oligosaccharide alditols that are dissociated under low-energy collision conditions using quadrupole orthogonal time-offlight (QoTOF) instrumentation and up to pseudo MS 3 mass spectrometry. Glycoforms, including isomers, are readily identified and their structures assigned. The isomer-specific spectra include highly informative cross-ring and elimination fragments, branch position specific signatures, and glycosidic bond fragments, thus facilitating linkage, branch, and sequence assignment. The method is sensitive and can be applied using as little as 40 fmol of derivatized oligosaccharide. Because permethylation renders oligosaccharides nearly chemically equivalent in the mass spectrometer, the method is semiquantitative and, in this regard, is comparable to methods reported using high field NMR and capillary electrophoresis. In this postgenomic age, the importance of glycosylation in biological processes has become clear. The nature of many of the important questions in glycomics is such that sample material is often extremely limited, thus necessitating the development of highly sensitive methods for rigorous structural assignment of the oligosaccharides in complex mixtures. The glycomics platform presented here fulfills these criteria and should lead to more facile glycomics analyses. (J Am

“…In addition, MALDI analyses suffer from matrix-dependent ionization and fragmentation processes [24 -27]. For instance, the "hot" matrix, ␣-cyano 4-hydroxycinnamic acid (CHCA), commonly used with peptides and small neutral glycans, is known to promote formation of glycosidic cleavages, whereas 2,5-dihydroxybenzoic acid (DHB), a "cooler" matrix that is typically used for glycopeptides and glycans, is said to suppress MALDI-induced fragmentation [24]. As a result, the type of matrix used for glycopeptide analysis largely influences the extent and type of fragmentation ions produced during MALDI-MS/MS experiments [26].…”

mentioning

confidence: 99%

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Irungu

Zhang

et al. 2008

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140⌬CFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis. (J