Differentiation of isomeric oligosaccharide structures by ESI tandem MS and GC-MS

A mixture of arabinoxylan oligosaccharides from wheat seedling was permethylated and analyzed by electrospray ion trap MS and GC-MS. The presence of isomeric structures differing in degree of branching and position of the branched residue along the xylose backbone was demonstrated for oligosaccharides with four and five monosaccharide residues. No isomeric structures were found for oligosaccharides with three monosaccharide residues. Linkage analysis by GC-MS reveals that xylose residues were substituted with single arabinoxyl residues at C-3.     

Glycomic strategy for efficient linkage analysis of di-, oligo- and polysialic acids

Sialic acid polymers of glycoproteins and glycolipids are characterized by a high diversity in nature and are involved in distinct biological processes depending inter alia on the glycosidic linkages between the present sialic acid residues. Though suitable protocols are available for chain length and sialic acid determination, sensitive methods for linkage analysis of di-, oligo-, and polysialic acids (di/oligo/polySia) are still pending. In this study, we have established a highly sensitive glycomic strategy for this purpose which is based on permethylation of di/oligo/polySia after tagging their reducing ends with the fluorescent dye 1,2-diamino-4,5-methylenedioxybenzene (DMB). Using DMB-labeled sialic acid di/oligo/polymers glycosidic linkages could be efficiently determined and, optionally, the established working procedure can be combined with HPLC for in depth characterization of distinct di/oligo/polySia chains. Moreover, the outlined approach can be directly applied to mammalian tissue samples and linkage analysis of sialic acid polymers present in biopsy samples of neuroblastoma tissue demonstrating the usefulness of the outlined work flow to screen, for example, cancer tissue for the presence of distinct variants of di/oligo/polySia as potentially novel biomarkers. Hence, the described strategy offers a highly sensitive and efficient strategy for identification of glycosidic linkages in sialic acid di/oligo/polymers of glycoproteins and glycolipids.     

Serum N-glycan and O-glycan analysis by mass spectrometry for diagnosis of congenital disorders of glycosylation

Congenital disorders of glycosylation (CDGs) are caused by defects in genes that participate in biosynthetic glycosylation pathways. To date, 19 different genetic defects in N-glycosylation, 17 in O-glycosylation, and 21 in multiple glycosylation are known. Current diagnostic testing of CDGs largely relies on indirect analysis of glycosylation of serum transferrin. Such analysis alone is insufficient to diagnose many of the known glycosylation disorders. To improve the diagnosis of these groups of CDGs, we have developed serum or plasma N- and O-glycan profiling using a combination of MALDI–TOF/MS and LC–MS/MS technologies. Using this approach, we analyzed samples from nine patients with different known multiple glycosylation disorders, including three with COG deficiencies, one with TMEM165-CDG, two with PGM1-CDG, and three with SLC35A2-CDG, and one patient with combined type I and type II of unknown molecular etiology. Measurement of the relative quantities of various N- and O-glycan species clearly differentiates patients and controls. Our study demonstrates that structural analysis and quantitation of combined N- and O-glycan profiles are reliable diagnostic tools for CDGs.     

An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin

Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)–TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006–2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.     

Glycomic profiling of invasive and non-invasive breast cancer cells

Quantitative profiling of glycans with different structures appears essential for a better understanding of the cellular adhesion phenomena associated with malignant transformation and the underlying aberrant glycosylation of cancer cells. Using the recently developed glycomic techniques and mass-spectrometric measurements, we compare the N-linked and O-linked oligosaccharide profiles for different breast cancer cell lines with those of normal epithelial cells. Statistically significant differences in certain neutral, sialylated and fucosylated structures are readily discerned through quantitative measurements, indicating a potential of distinguishing invasive and non-invasive cancer attributes. The glycomic profile data cluster accordingly using Principal Component Analysis, verifying further glycobiological differences due to the differences between normal and cancer cell lines.     

Effect of acetylation and permethylation on the conformation and candidacidal activity of salivary histatin-5

Summary Salivary histatins provide the non-immune defense against oral pathogens such as Candida albicans. The structural requirements of histatin-5 for anticandida activity were examined with respect to its ability to adopt helical structures, its electrostatic interactions and the hydrogen-bonding potency of its basic residues. For this purpose, the lysine and/or histidine residues of histatin-5 were chemically modified by acetylation and permethylation. Acetylated histatin-5 retained its ability to adopt helical structures in 2,2,2-trifluoroethanol, but completely lost its ability to kill yeast cells. In contrast, permethylated histatin-5 shows very little tendency to adopt a helical structure, but retained significant anticandida activity. The results suggest that the candidacidal activity can arise even when the histatin does not have the ability to adopt helical structures. The candidacidal activity of the derivatives is discussed in terms of electrostatic interactions and hydrogen-bonding potency.     

Model studies on methyl amyloses: correlation between reaction conditions and primary structure

Methyl amyloses have been prepared under various conditions and studied as model compounds for the determination of the substitution pattern in the polymer chain. After permethylation with iodomethane-d₃ the glucosidic linkages were statistically cleaved by partial methanolysis or reductive cleavage. The distribution of substituents in the dimer-, trimer-, and tetramer fraction was determined by FAB-MS and MALDI-TOF-MS and compared with that calculated from the monomer composition. While a homogeneous methylation in water gave the expected random distribution, a reaction in Me₂SO solution with sodium hydroxide and iodomethane yielded a methyl amylose with a surprising bimodal substitution pattern. A third example indicates a ds gradient in the sample as a result of topochemical reaction control.