Linkage and branch determination of N-linked oligosaccharides using sequential degradation/closed-ring chromophore labeling/negative ion trap mass spectrometry

Abstract

A method based on sequential degradation, p-aminobenzoic ethyl ester (ABEE) closed-ring labeling, and negative ion electrospray ionization tandem mass spectrometry is presented for the study of linkage and branch determination for N-linked oligosaccharides. Closed-ring labeling provides greater linkage information than the more popular open-ring reductive amination approach. In addition, after high-performance liquid chromatography (HPLC) separation, closed-ring labeling allows for regeneration of the underivatized oligosaccharide, a requirement for alkaline sequential degradation. The analytical scheme presented here uses HPLC separation of closed-ring labeled oligosaccharides to resolve the mixture into individual forms that undergo subsequent structural analysis by negative ion tandem mass spectrometry. To facilitate complete structural analysis, particularly for larger sugars, the closed-ring labels are removed and the sugars are sequentially degraded by controlled alkaline hydrolysis. It is noteworthy that for sugars containing sialic acid moieties, a protecting group must be used to stabilize sialic acid groups during sequential alkaline degradation. This described approach was applied to two high mannose oligosaccharides M5G2, M6G2 cleaved from the ribonuclease B and a complex oligosaccharide A2 cleaved from transferrin. T he amino acid sequence alone is generally insufficient to describe protein function. The biologically active forms of mature proteins are often post-translationally modified and these modifications typically influence protein function. Glycosylation is a predominant modification mechanism. It has been estimated that 60 -90% of all mammalian proteins are glycosylated [1]. The carbohydrate chains play critical roles in numerous biological processes, including fertilization, immune response, viral replication, parasitic inflection, cell-cell adherence, degradation of blood clots, and inflammation, for example [1][2][3][4]. The structural diversity inherent in the sugar moieties of a glycoprotein enables subtle changes in protein shape, charge, and volume, which can affect function both temporally and spatially. To achieve a full understanding of the function of a glycoprotein, a detailed characterization of its glycan structure is imperative.Structural elucidation of complex carbohydrates requires determination of monosaccharide composition, sequence, branching pattern, glycosidic linkages, and anomeric configuration. Nuclear magnetic resonance (NMR) comes close to providing complete structural analysis, although the amount of sample required and difficulty with analyzing complex mixtures limits the utility of NMR, particularly for proteomics applications. In recent years, mass spectrometry (MS) has become a key tool for structural analysis of carbohydrates. Several mass spectrometric techniques have been proved to be useful analytical tools for the determination of oligosaccharide structure. All of these techniques have the advantages over traditional methods [5], such as low sample consu...

Alkali‐hydroxide‐doped matrices for structural characterization of neutral underivatized oligosaccharides by MALDI time‐of‐flight mass spectrometry

“…The method of identifying the reducing end residue by partial alkaline degradation is simple and straightforward, requiring no time‐consuming derivatization such as reductive amination and methylation 11–16. Additionally, it reduces the degree of complexity in sequencing oligosaccharides owing to the loss of one component sugar.…”

“…The observation is explained in terms of the fact that neutral underivatized oligosaccharides have a low affinity for protons but yet they can be multiply coordinated to alkali metal cations through electrostatic interactions 7–9. Consequently, chemical derivatizations such as ligation to a peptide10 or labeling with an ionizable group11–16 have been frequently carried out to enhance the proton affinity, and thus, ionization efficiency of the analyte. The derivatization, while time‐consuming and unavoidably resulting in sample loss, offers an additional advantage—it leads to enhanced and well defined fragmentation patterns in tandem MS analysis and, thereby, allows straightforward identification of the sugar residue at the reducing end of the oligosaccharide.…”

Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry

“…Schiff bases can exist in equilibrium with the closed ring form of the sugar and have been assumed to be in this form in studies by Cheng et al [121,122]. The derivatives with ethyl-p-aminobenzoate (ABEE, 13) gave good HPLC characteristics and structural information was obtained by negative ion electrospray MS.…”

Sequencing and characterization of oligosaccharides using infrared multiphoton dissociation and boronic acid derivatization in a quadrupole ion trap

“…While it has become common practice to form chromophore-labeled carbohydrate derivatives for UV and fluorescence detection following chromatographic separation, various chemical derivatization strategies of oligosaccharides have also been used in conjunction with mass spectrometry to increase ionization sensitivity as well as yield more informative fragmentation patterns that reveal linkage and anomeric configuration [52][53][54][55][56][57][58]. Some of the derivatization techniques used for ESI-MS analysis of carbohydrates include permethylation for linkage information [52,53], reductive amination for attachment of chromophores for HPLC detection [54], p-aminobenzoic ethyl ester derivatization via formation of a glycosylamine for determination of linkage and branching information [55], and modification of cis-diol groups of disaccharides with boronic acids for stereochemical determination [56,57].…”

“…Some of the derivatization techniques used for ESI-MS analysis of carbohydrates include permethylation for linkage information [52,53], reductive amination for attachment of chromophores for HPLC detection [54], p-aminobenzoic ethyl ester derivatization via formation of a glycosylamine for determination of linkage and branching information [55], and modification of cis-diol groups of disaccharides with boronic acids for stereochemical determination [56,57]. Most recently, phenylboronic acid was used as a reagent ion for the analysis of biologically active diol compounds by desorption electrospray ionization (DESI) MS [58].…”