Glycosylation is a common post-translational modification to cell surface and extracellular matrix (ECM) proteins as well as to lipids. As a result, cells carry a dense coat of carbohydrates on their surfaces that mediates a wide variety of cell-cell and cell-matrix interactions that are crucial to development and function. Because of the historical difficulties with the analysis of complex carbohydrate structures, a detailed understanding of their roles in biology has been slow to develop. Just as mass spectrometry has proven to be the core technology behind proteomics, it stands to play a similar role in the study of functional implications of carbohydrate expression, known as glycomics. This review summarizes the state of knowledge for the mass spectrometric analysis of oligosaccharides with regard to neutral, sialylated, and sulfated compound classes. Mass spectrometric techniques for the ionization and fragmentation of oligosaccharides are discussed so as to give the reader the background to make informed decisions to solve structure-activity relations in glycomics.

SUMMARY Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.

The biological significance of protein and lipid glycosylation is well established. For example, cells respond to environmental stimuli by altering glycan structures on their surfaces, and cancer cells evade normal growth regulation in part by remodeling their surface glycans. In general, glycan chemical properties differ significantly from those of proteins, lipids, nucleic acids and small molecule metabolites. Thus, advances in glycomics, a comprehensive study to identify all glycans in an organism, rely on the development of specialized analytical methods. Mass spectrometry (MS) is emerging as an enabling technology in the field of glycomics. This review summarizes recent developments in mass spectrometric analysis methods for protein-based glycomics and glycoproteomics workflows.

No abstract

Glycosylation defines the adhesive properties of animal cell surfaces and the surrounding extracellular environments. Because cells respond to stimuli by altering glycan expression, glycan structures vary according to spatial location in tissue and temporal factors. These dynamic structural expression patterns, combined with the essential roles glycans play in physiology, drive the need for analytical methods for glycoconjugates. In addition, recombinant glycoprotein drug products represent a multibillion dollar market. Effective analytical methods are needed to speed the identification of new targets and the development of industrial glycoprotein products, both new and biosimilar. Mass spectrometry is an enabling technology in glycomics. This review summarizes mass spectrometry of glycoconjugate glycans. The intent is to summarize appropriate methods for glycans given their chemical properties as distinct from those of proteins, lipids, and small molecule metabolites. Special attention is given to the uses of mass spectral profiling for glycomics with respect to the N-linked, O-linked, ganglioside, and glycosaminoglycan compound classes. Next, the uses of tandem mass spectrometry of glycans are summarized. The review finishes with an update on mass spectral glycoproteomics.

Summary Most proteins are glycosylated. Mass spectrometry methods are used for mapping glycoprotein glycosylation and detailed glycan structural determination. This technology enables precise characterization of recombinant glycoproteins in the pharmaceutical industry and academic biomedicine.

The structural characterization of heparin-like glycosaminoglycans (HLGAGs) is a major challenge in glycobiology. These linear, sulfated oligosaccharides are expressed on animal cell surfaces, in extracellular matrixes, basement membranes, and mast cell granules and bind with varying degrees of specificity to families of proteases, growth factors, chemokines, and blood coagulation proteins. Cell surface HLGAGs bind growth factors and growth factor receptors and serve as coreceptors in these interactions. Understanding of the mechanism and regulation of growth factor-receptor binding requires efficient determination of cell surface HLGAG structures and the variations in their expression in response to the cellular environment. The solution to this problem entails rapid, sensitive structural analysis of these molecules. To date, HLGAG sequencing requires multistep processes that combine chemical and enzymatic degradation with gel-based or mass spectrometry-based detection systems. Although tandem mass spectrometry has revolutionized proteomics, the fragility of sulfate groups has limited its usefulness in the analysis of HLGAGs. This work demonstrates that tandem mass spectrometry can be effectively used to determine HLGAG structures while minimizing losses of SO3. First, collision-induced dissociation (CID) is shown to produce abundant backbone cleavage ions for HLGAG oligosaccharides, provided that most sulfate groups are deprotonated. Fragmentation of different precursor ion charge states produces complementary data on the structure of the HLGAG. Second, calcium ion complexation of HLGAGs stabilizes the sulfate groups, increases the relative abundances of backbone cleavage ions, and decreases the abundances of ions produced from SO3 losses.

ABSTRACT