Abstract
The fact that practically all living cells are covered with carbohydrates gives an indication that these unique molecules are of tremendous importance to life with impacts in health and disease. One example of the significance of carbohydrates is found in the blood types that are determined by minor differences in the structure of the glycans on the surface of red blood cells. Other examples include glycosylation changes in cancer, the masking of viruses by glycans to evade the host immune response, and the inflammation resulting from exposure to endotoxins in gram-negative bacteria, among many others. As science delves deeper into understanding biological processes, it is increasingly confronted with the challenge to account for the roles that carbohydrates play in the molecular interactions that drive these processes. Molecular interactions are based on molecular shape, and molecular shape is determined by primary structure. Thus, it is vital to enlarge our repertoire of analytical tools for the elucidation of the primary structure of carbohydrates. Although a number of traditional analytical tools, including monosaccharide and linkage analysis by gas chromatography/mass spectrometry (GC/MS), glycan composition analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), sequence analysis by tandem mass spectrometry (MSn), and nuclear magnetic resonance (NMR) spectroscopy, have been available for some time and have been used with tremendous success, a host of challenges posed by the specific structural peculiarities of carbohydrates remain to be met. These include the varying stability of glycosidic bonds and free monosaccharides to hydrolysis, the nonstoichiometric substitution by noncarbohydrate residues, the difficulty of automating the existing protocols for high-throughput analysis, the scarcity of standards, the unparalleled structural diversity and close similarity of different structures, and the lack of chromophores that can often not be directly overcome with labeling.
The articles in this special issue are a sampling of ingenious ways in which scientists have dealt with these challenges. The special issue is divided into three conceptually distinct parts, monosaccharide analysis, glycomics, and glycan–protein interactions.
Monosaccharide Analysis
This section begins with Jeff Rohrer’s review 1 on the quantification of mono- and oligosaccharides produced by complete or partial depolymerization of carbohydrate conjugate vaccines using high-performance anion-exchange chromatography (HPAEC). It is important for effective quality control to be able to accurately and consistently measure the level of conjugation of capsular polysaccharides to the carrier protein. Two papers in this section describe innovative methods to determine the location and degree of methylation in plant pectic polysaccharides. O’Neill et al. 2 show how reduction and isotopic labeling can be used to detect minor quantities of galacturonic acid O-methyl ethers and glucuronic acid methyl esters in rhamnogalacturonan II of several plant species with a view to understand the role of borate-mediated dimerization. Smith et al. 3 present analytical methods for the quantification of 4-O-methylglucuronic acid in glucuronoxylan and arabinogalactan and show their application in studying the function of plant cell wall synthesis enzymes. Hung et al. 4 propose a monosaccharide derivatization method based on the reaction of reducing sugars with naphthimidazole, which forms single derivatives that confer both chromatographic retention and fluorescence for detection. The reagent labels both aldoses and α-keto acids, and the method utilizes flow reactors, priming it for automation.
Glycomics
The second section of this special issue is devoted to glycomics, which is the comprehensive analysis of glycans from glycoproteins, highlighting advances toward high throughput through automation and improvements benefiting the preparation of glycan arrays. Shajahan et al. 5 developed a high-throughput method for permethylation of released glycans and propose an automated MSn workflow for the rapid structure characterization of glycoproteins. Zhang et al. 6 likewise put forth an efficient high-throughput method for glycomics analysis featuring automated sample preparation, rapid liquid chromatography with fluorescence and mass spectrometric detection, and MS-based target screening for quality control of therapeutic antibody N-glycans. Zhang et al. 7 developed O-benzylhydroxylamine (BHA) as a cleavable tag that is both hydrophobic for chromatographic separation of glycans and chromogenic for detection of glycans, in order to obtain sufficient amounts of glycans for functional studies using glycan arrays.
Glycan–Protein Interactions
The last section of this special issue deals with glycan–protein interactions, which require prior knowledge of primary structures and so are a step closer to functional studies of carbohydrates. Vignovich and Pomin 8 review saturation difference (STD) NMR spectroscopy to study the interactions of glycosaminoglycans with proteins. STD specifically allows the determination of which parts of the ligand interact with the protein, which is significant for the study of structure–activity relationships. Bi et al. 9 present a detailed description of the preparation and application of a nanosensor for the sensitive detection of oversulfated chondroitin sulfate (OSCS) in heparin. This sensor works because OSCS disrupts the binding between heparin lyase enzyme and heparin and is more sensitive than any other method for OSCS detection to date.