Glycoprotein Constituents Research Articles

Central Role of β-1,4-GalT-V in Cancer Signaling, Inflammation, and Other Disease-Centric Pathways.

UDP-Galactose: Glucosylceramide, β-1,4-Galactose transferase-V (β-1,4-GalT-V), is a member of a large glycosyltransferase family, primarily involved in the transfer of sugar residues from nucleotide sugars, such as galactose, glucose mannose, etc., to sugar constituents of glycosphingolipids and glycoproteins. For example, UDP-Galactose: Glucosylceramide, β-1,4-galactosyltransferase (β-1,4-GalT-V), transfers galactose to glucosylceramide to generate Lactosylceramide (LacCer), a bioactive "lipid second messenger" that can activate nicotinamide adenine dinucleotide phosphate(NADPH) oxidase (NOX-1) to produce superoxide's (O2-) to activate several signaling pathways critical in regulating multiple phenotypes implicated in health and diseases. LacCer can also activate cytosolic phospholipase A-2 to produce eicosanoids and prostaglandins to induce inflammatory pathways. However, the lack of regulation of β-1,4-GalT-V contributes to critical phenotypes central to cancer and cardiovascular diseases, e.g., cell proliferation, migration, angiogenesis, phagocytosis, and apoptosis. Additionally, inflammation that accompanies β-1,4-GalT-V dysregulation accelerates the initiation and progression of cancer, cardiovascular diseases, as well as inflammation-centric diseases, like lupus erythematosus, chronic obstructive pulmonary disease (COPD), and inflammatory bowel diseases. An exciting development in this field of research arrived due to the recognition that the activation of β-1,4-GalT-V is a "pivotal" point of convergence for multiple signaling pathways initiated by physiologically relevant molecules, e.g., growth factors, oxidized-low density lipoprotein(ox- LDL), pro-inflammatory molecules, oxidative and sheer stress, diet, and cigarette smoking. Thus, dysregulation of these pathways may well contribute to cancer, heart disease, skin diseases, and several inflammation-centric diseases in experimental animal models of human diseases and in humans. These observations have been described under post-transcriptional modifications of β-1,4- GalT-V. On the other hand, we also point to the important role of β-1-4 GalT-V-mediated glycosylation in altering the formation of glycosylated precursor forms of proteins and their activation, e.g., β-1 integrin, wingless-related integration site (Wnt)/-β catenin, Frizzled-1, and Notch1. Such alterations in glycosylation may influence cell differentiation, angiogenesis, diminished basement membrane architecture, tissue remodeling, infiltrative growth, and metastasis in human colorectal cancers and breast cancer stem cells. We also discuss Online Mendelian Inheritance in Man (OMIM), which is a comprehensive database of human genes and genetic disorders used to provide information on the genetic basis of inherited diseases and traits and information about the molecular pathways and biological processes that underlie human physiology. We describe cancer genes interacting with the β-1,4-GalT-V gene and homologs generated by OMIM. In sum, we propose that β-1,4-GalT-V gene/protein serves as a "gateway" regulating several signal transduction pathways in oxidative stress and inflammation leading to cancer and other diseases, thus rationalizing further studies to better understand the genetic regulation and interaction of β-1,4-GalT-V with other genes. Novel therapies will hinge on biochemical analysis and characterization of β-1,4-GalT-V in patient-derived materials and animal models. And using β-1,4-GalT-V as a "bonafide drug target" to mitigate these diseases.

Structure and Role of O-Linked Glycans in Viral Envelope Proteins.

N- and O-glycans are both important constituents of viral envelope glycoproteins. O-linked glycosylation can be initiated by any of 20 different human polypeptide O-acetylgalactosaminyl transferases, resulting in an important functional O-glycan heterogeneity. O-glycans are organized as solitary glycans or in clusters of multiple glycans forming mucin-like domains. They are functional both in the viral life cycle and in viral colonization of their host. Negatively charged O-glycans are crucial for the interactions between glycosaminoglycan-binding viruses and their host. A novel mechanism, based on controlled electrostatic repulsion, explains how such viruses solve the conflict between optimized viral attachment to target cells and efficient egress of progeny virus. Conserved solitary O-glycans appear important for viral uptake in target cells by contributing to viral envelope fusion. Dual roles of viral O-glycans in the host B cell immune response, either epitope blocking or epitope promoting, may be exploitable for vaccine development. Finally, specific virus-induced O-glycans may be involved in viremic spread.

One-Pot Glycosylation Strategy Assisted by Ion Mobility-Mass Spectrometry Analysis toward the Synthesis of N-Linked Oligosaccharides.

N-Glycans are major constituents of several cellular glycoproteins. One-pot strategies for the synthesis of N-glycans are crucial for the rapid generation of pure samples to determine their biological functions. Herein, we describe a double one-pot strategy for the synthesis of N-glycans assisted by an IM-MS analysis approach for rapid screening of optimized glycosylation reaction conditions. This research includes triflate-mediated direct β-mannosylation and tandem glycosylation in a one-pot strategy for the synthesis of the challenging N-linked trisaccharide core β-5. Furthermore, a one-pot sequential glycosylation of the N-linked trisaccharide core 7 furnishes diverse high-mannose type N-glycans with excellent stereo- and regioselectivities. In particular, ion mobility-mass spectrometry-based quantitative analysis is applied to identify the stereo- and regioselective outcomes of the crude reaction mixtures to develop a highly efficient one-pot protocol.

Polyglycerol-Based Mucus-Inspired Hydrogels.

The mucus layer is a hydrogel network that covers mucosal surfaces of the human body. Mucus has important protective properties that are related to its unique rheological properties, which are based on mucins being the main glycoprotein constituents. Mucin macromolecules entangle with one another and form a physical network that is instrumental for many important defense functions. Mucus derived from various human or animal sources is poorly defined and thus not suitable for many application purposes. Herein, a synthetic route is fabricated to afford a library of compositionally defined mucus-inspired hydrogels (MIHs). MIHs are synthesized by thiol oxidation to render disulfide bonds between the crosslinker ethoxylated trimethylolpropane tri(3-mercaptopropionate) (THIOCURE ETTMP 1300) and the linear precursors, dithiolated linear polyglycerol (LPG(SH)2 ) or polyethylene glycol (PEG(SH)2 ) of different molecular weights. The mixing ratio of linear polymers versus crosslinker and the length of the linear polymer are varied, thus delivering a library of compositionally defined mucin-inspired constructs. Their viscoelastic properties are determined by frequency sweeps at 25 and 37 °C and compared to the corresponding behavior of native human mucus. Here, MIHs composed of a 10:1 ratio of LPG(SH)2 and ETTMP 1300 are proved to be the best comparable to human airway mucus rheology.

Arabinose biosynthesis is critical for salt stress tolerance in Arabidopsis.

The capability to maintain cell wall integrity is critical for plants to adapt to unfavourable conditions. l-Arabinose (Ara) is a constituent of several cell wall polysaccharides and many cell wall-localised glycoproteins, but so far the contribution of Ara metabolism to abiotic stress tolerance is still poorly understood. Here, we report that mutations in the MUR4 (also known as HSR8) gene, which is required for the biosynthesis of UDP-Arap in Arabidopsis, led to reduced root elongation under high concentrations of NaCl, KCl, NaNO3 , or KNO3 . The short root phenotype of the mur4/hsr8 mutants under high salinity is rescued by exogenous Ara or gum arabic, a commercial product of arabinogalactan proteins (AGPs) from Acacia senegal. Mutation of the MUR4 gene led to abnormal cell-cell adhesion under salt stress. MUR4 forms either a homodimer or heterodimers with its isoforms. Analysis of the higher order mutants of MUR4 with its three paralogues, MURL, DUR, MEE25, reveals that the paralogues of MUR4 also contribute to the biosynthesis of UDP-Ara and are critical for root elongation. Taken together, our work revealed the importance of the Ara metabolism in salt stress tolerance and also provides new insights into the enzymes involved in the UDP-Ara biosynthesis in plants.

Plasma mannose level, a putative indicator of glycogenolysis, and glucose tolerance in Japanese individuals.

Aims/IntroductionMannose is a monosaccharide constituent of glycoproteins and glycolipids. Experiments in rats have shown previously that the plasma mannose level decreases after glucose load, but does not decrease in diabetic rats, and that hepatic glycogenolysis is a source of this plasma mannose; however, these results are not fully elucidated in humans. Plasma mannose levels before/after glucose loading in humans with various degrees of glucose intolerance were examined to analyze their association with clinical factors.Materials and MethodsThe 75‐g oral glucose tolerance test was carried out in Japanese individuals not taking diabetes medications. Participants were classified into normal glucose tolerance, impaired glucose metabolism and diabetes mellitus groups. Insulinogenic index as an index of insulin secretion, and Matsuda Index as an index of insulin sensitivity were calculated. Mannose was assayed by the established method using high‐performance liquid chromatography after labeling.ResultsAfter glucose load, the plasma mannose level decreased gradually in the normal glucose tolerance group, but did not decrease in the diabetes mellitus group. Plasma mannose changes during 120 min from baseline (M120‐M0) were significantly different among the three groups (normal glucose tolerance: −16.7 ± 1.7; impaired glucose metabolism: −9.0 ± 1.9; diabetes mellitus: −1.4 ± 1.8 μmol/L [n = 25 in each group], P < 0.0001). Plasma glucose 120 min after glucose loading (R 2 = 0.412) or loge‐insulinogenic index, loge‐Matsuda Index and age (R 2 = 0.230) were determinants of M120‐M0 in multiple regression analyses.ConclusionsWe clarified the relationship between plasma mannose level and glucose tolerance in humans. The present results are compatible with those using rats, in which mannose derived from glycogenolysis plays an important role in the alteration of mannose levels after glucose loading.

Synthesis of (2R)- and (2S)-aminooxy analogues of β-O-glucosylserine andN-oxyamide linked glycoconjugates

Glycosyl amino acids are constituents of glycopeptides and glycoproteins playing important roles in various cellular activities and pathogenic processes. We have stereoselectively synthesized both (2R)- and (2S)-aminooxy analogues of β-O-glucosylserine from sn-(3-β-O-glucosyl)glycerol through selective protection, Mitsunobu reaction, oxidation and Lattrell-Dax epimerization reactions. N-oxyamide linked glycopetides and glycolipids have been prepared from the β-O-glucosyl aminooxy acid derivatives. 1H NMR and IR studies suggest the formation of an α N−O turn structure in the glycopeptide 19.

Synthesis of C-Glucosyl Aminooxy Esters

Glycosyl amino acids are constituents of glycopeptides and glycoproteins playing key roles in biological processes. We have synthesized C -glucosyl aminooxy acid derivatives as novel glycosyl amino acid building blocks for the generation of glycopeptide and glycoprotein mimics. These compounds can be readily prepared from C -allyl glucosides, through dihydroxylation, selective tritylation, Mitsunobu reaction with PhthNOH, detritylation, and oxidation. Both C -glucosyl l - and d -α-aminooxy esters have been successfully synthesized. With the ready availability of C -allyl glycosides, the present methodology could be applied to the synthesis of other C -glycosyl aminooxy acid derivatives.

Milk 3′ sialyllactose does not increase brain sialic acid concentration in mice

Sialic acid (Sia), a carboxylated 9‐carbon monosaccharide, is a crucial constituent of brain glycoproteins and glycolipids associated with brain development and enhanced cognitive function. Brain Sia concentrations rapidly increase after birth and are significantly higher in breast‐fed than in formula‐fed infants. Compared to formula, human breast milk contains a high amount of sialylated oligosaccharides; yet their direct role in brain Sia accrual is unknown. Our objective was to determine whether sialylated milk oligosaccharides contribute to brain Sia accumulation in mice between birth and weaning. To overcome problems with artificial feeding in newborn pups, we used a cross‐foster approach with pups reared on dams normal or genetically deficient in 3′sialyllactose (3′SL), one of the major sialylated milk oligosaccharides. Urine 3′SL and brain Sia were derivatized and measured by HPLC. While pups reared on dams with normal milk 3′SL had significantly higher urine 3′SL levels than pups fed on 3′SL deficient milk (460 vs. 40 uM at day 7, P&lt;0.001), there was no significant difference in brain Sia one week (6.0±1.1 vs. 6.0±1.2 pmol/ug protein) and three weeks after birth (19.0±9.7 vs. 15.0±4.9 pmol/ug protein). The data indicate that 3′SL, one of the major sialylated milk oligosaccharides, does not contribute to postnatal brain Sia accumulation – at least in mice.Grant Funding Source: NIH

Inositolphosphoceramide Metabolism in Trypanosoma cruzi as Compared with other Trypanosomatids

Chagas disease is caused by Trypanosoma cruzi and is endemic to North, Central and South American countries. Current therapy against this disease is only partially effective and produces adverse side effects. Studies on the metabolic pathways of T. cruzi, in particular those with no equivalent in mammalian cells, might identify targets for the development of new drugs. Ceramide is metabolized to inositolphosphoceramide (IPC) in T. cruzi and other kinetoplastid protists whereas in mammals it is mainly incorporated into sphingomyelin. In T. cruzi, in contrast to Trypanosoma brucei and Leishmania spp., IPC functions as lipid anchor constituent of glycoproteins and free glycosylinositolphospholipids (GIPLs). Inhibition of IPC and GIPLs biosynthesis impairs differentiation of trypomastigotes into the intracellular amastigote forms. The gene encoding IPC synthase in T. cruzi has been identified and the enzyme has been expressed in a cell-free system. The enzyme involved in IPC degradation and the remodelases responsible for the incorporation of ceramide into free GIPLs or into the glycosylphosphatidylinositols anchoring glycoproteins, and in fatty acid modifications of these molecules of T. cruzi have been understudied. Inositolphosphoceramide metabolism and remodeling could be exploited as targets for Chagas disease chemotherapy.

EFFECT OF OVERIECTOMY AND OF ESTROGEN ADMINISTRATION UPON DUODENAL ULCERATION INDUCED BY CYSTEAMINE

Duodenal ulcers were induced in ovariectomized, intact and old mice using cysteamine hydrochloride. Under these experimental conditions ovariectomy and old age strikingly increased sensitivity to ulcer induction while estrogen administration showed a decrease in sensitivity to ulcer induction. Nevertheless, the administration of estrogen in old mice showed no effects in either intact or overiectomized mice. This change in ulcer sensitivity reflected from histological, histochemical and biochemical studies. The histological study was performed by using haematoxylin-eosin and haematoxyline staining technique. The histochemistry of the duodenal region was studied by using periodic acid Schiff reaction (PAS) for glycoproteins. The biochemical study was performed to study various constituents of glycoproteins like hexose, fucose, sialic acid and the protein. The result showed that ulcer severity was more in overiectomized cysteamine treated mice and old mice treated with cysteamine. The histological studies showed that ovariectomy decreased or did not show any change in the ulcer sensitivity considering cryptus Lieburkuhn and Brunner's glands. The same result reflected by differential intensity in the staining property of the Brunner's gland. The biochemical study showed that the glycoprotein contents were reduced many times in overiectomized cysteamine treated mice and their reversal in estrogen administered ovariectomized cysteamine injected mice. These findings prove that estrogen protects the duodenal ulcer from cysteamine administration.

Highly Alpha‐Selective Sialyl Phosphate Donors for Efficient Preparation of Natural Sialosides

Hubble, bubble, toil, and trouble: The use of a new sialyl phosphate donor allows the stereoselective, one-pot multicomponent synthesis of α-sialooligosaccharides (see scheme).

Enzymatic activity of alpha-L-fucosidase and L-fucokinase across vertebrate animal species.

The oligosaccharide portion of glycoproteins is known to modulate protein structure, function, and turnover. Our laboratory is interested in the metabolism of L-fucose, a normal constituent of eukaryotic glycoproteins. L-fucose is unique in that it is the only levorotatory sugar utilized in mammalian systems. There is considerable interest in understanding the controls which determine the level of L-fucose attached to proteins, in order to generate stable and active glycoforms of protein for the treatment of disease. As part of a program to determine the controls on protein L-fucosylation, we have systematically determined the tissue distribution of the enzymes L-fucokinase and alpha-L-fucosidase in species across the vertebrate animal kingdom. In general, the level of alpha-L-fucosidase is higher than L-fucokinase level. The tissue with highest enzyme activity cannot be generalized, regardless of which enzyme is of interest. Furthermore, there is not a correlation between synthetic and catabolic enzyme activity within a tissue. L-fucokinase can be detected in all tissues examined. Interestingly, we have also detected ss-D-fucosidase activity, present in extraordinary levels in the liver and small intestine of snake. Whether this is due to a specific enzyme or whether it represents a broad specificity of the alpha-L-fucosidase is currently being investigated.

Small molecule probes of mycobacterial cell wall assembly

Carbohydrates mediate fundamental processes in humans; they also can be essential for pathogen survival. For example, oligosaccharides serve as essential components of the mycobacterial cell wall. Agents that inhibit key biosynthetic steps in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), would be valuable, because TB results in approximately 2 million deaths/year. A key component of the mycobacterial cell wall is the arabinogalactan polysaccharide, which contains galactofuranose (Galf) residues. Because Galf residues are not constituents of human glycoproteins or glycolipids, we have focused on understanding galactofuranose incorporation. To this end, we have discovered a fundamentally new catalytic role for the heterocyclic cofactor flavin (vitamin B2), and we have found that an essential glycosyltransferase mediates oligosaccharide assembly by a processive mechanism. Moreover, we have discovered small molecule inhibitors of galactofuranose incorporation. Small molecules targeting this pathway block mycobacterial growth, a result that highlights the benefits of blocking this glycoconjugate biosynthetic pathway. We anticipate that these inhibitors can illuminate carbohydrate function and lead to the generation of therapeutics effective against strains resistant to current anti‐mycobacterial agents. This research was supported by the National Institute of Allergy and Infectious Diseases.

Egress of Light Particles among Filopodia on the Surface of Varicella-Zoster Virus-Infected Cells

Varicella-zoster virus (VZV) is renowned for its very low titer when grown in cultured cells. There remains no single explanation for the low infectivity. In this study, viral particles on the surfaces of infected cells were examined by several imaging technologies. Few surface particles were detected at 48 h postinfection (hpi), but numerous particles were observed at 72 and 96 hpi. At 72 hpi, 75% of the particles resembled light (L) particles, i.e., envelopes without capsids. By 96 hpi, 85% of all particles resembled L particles. Subsequently, the envelopes of complete virions and L particles were investigated to determine their glycoprotein constituents. Glycoproteins gE, gI, and gB were detected in the envelopes of both types of particles in similar numbers; i.e., there appeared to be no difference in the glycoprotein content of the L particles. The viral particles emerged onto the cell surface amid actin-based filopodia, which were present in abundance within viral highways. Viral particles were easily detected at the base of and along the exterior surfaces of the filopodia. VZV particles were not detected within filopodia. In short, these results demonstrate that VZV infection of cultured cells produces a larger proportion of aberrant coreless particles than has been seen with any other previously examined alphaherpesvirus. Further, these results suggested a major disassociation between capsid formation and envelopment as an explanation for the invariably low VZV titer in cultured cells.

Preliminary Study Showing the Relationship Between Platelet Fibronectin, Sialic Acid, and ADP-induced Aggregation Levels in Coronary Heart Disease

Several studies indicate that thrombosis plays an important role in the pathogenesis of coronary heart disease (CHD). Fibronectin is a multifunctional protein in plasma, other body fluids, and cell surface and plays an important role in platelet functions, including mediation of cell-cell and cell-surface interactions. Sialic acid is a regular constituent of glycoproteins and gangliozides in the outer cell membrane of mammalian cells. Therefore, the sialic acid content of platelets, which are characterized by their ability to aggregate with each other, can be important in leading to thrombus formation. In this study, platelet fibronectin, sialic acid-, and adenosine diphosphate (ADP)-induced platelet aggregation levels were determined in patients with CHD. Platelet sialic acid concentrations were determined by Warren's method. Platelet aggregation tests with ADP in platelet-rich plasma (PRP) were analyzed by use of an aggregometer. Platelet homogenate fibronectin levels were determined by ELISA. Total protein levels were determined by Lowry method. Our results indicate that, in patients with no vessel disease (patients with no obstructed vessel but suffering from chest pain, like angina pectoris) platelet fibronectin levels were significantly lower than the total of the other patients (patients with 1, 2, or 3 obstructed coronary vessels) (p<0.05). Sialic acid levels in patients with no vessel disease were significantly lower than the total of the patient group (p<0.05). There was significant (+) correlation between platelet aggregation, platelet fibronectin, platelet sialic acid, and severity of disease (p<0.05). Our preliminary findings suggest that, especially platelet fibronectin levels potentially represent a pathogenic factor for CHD.

Hepatic glycogen breakdown is implicated in the maintenance of plasma mannose concentration

D-mannose is an essential monosaccharide constituent of glycoproteins and glycolipids. However, it is unknown how plasma mannose is supplied. The aim of this study was to explore the source of plasma mannose. Oral administration of glucose resulted in a significant decrease of plasma mannose concentration after 20 min in fasted normal rats. However, in fasted type 2 diabetes model rats, plasma mannose concentrations that were higher compared with normal rats did not change after the administration of glucose. When insulin was administered intravenously to fed rats, it took longer for plasma mannose concentrations to decrease significantly in diabetic rats than in normal rats (20 and 5 min, respectively). Intravenous administration of epinephrine to fed normal rats increased the plasma mannose concentration, but this effect was negated by fasting or by administration of a glycogen phosphorylase inhibitor. Epinephrine increased mannose output from the perfused liver of fed rats, but this effect was negated in the presence of a glucose-6-phosphatase inhibitor. Epinephrine also increased the hepatic levels of hexose 6-phosphates, including mannose 6-phosphate. When either lactate alone or lactate plus alanine were administered as gluconeogenic substrates to fasted rats, the concentration of plasma mannose did not increase. When lactate was used to perfuse the liver of fasted rats, a decrease, rather than an increase, in mannose output was observed. These findings indicate that hepatic glycogen is a source of plasma mannose.

Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins

C-mannosylation of Trp residue is one of the most recently discovered types of glycosylation, but the identification of these mannosylated residues in proteins is rather tedious. In a previous paper [Biochemistry 42 (2003) 8342], it was reported that the complete analysis of all constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample in three different steps of gas chromatography/mass spectrometry of heptafluorobutyrate derivatives. It was observed that during the acid-catalyzed methanolysis step used for liberation of monosaccharide from classical O- and N-glycans, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively. These derivatives were stable to acid hydrolysis used for the liberation of amino acids. Since monosaccharide derivatives were also stabilized as heptafluorobutyrate derivatives of O-methyl-glycosides, it was suggested that C-mannosides of Trp residues could quantitatively be recovered. Based on the analyses of standard compounds, peptides and RNase 2 from human urine, we report that C(2)-mannosylated Trp could be quantitatively recovered and identified during the step of amino acid analysis. Analyses of different samples indicated that this type of glycosylation is absent in bacteria and yeasts.

Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins

C-mannosylation of Trp residue is one of the most recently discovered types of glycosylation, but the identification of these mannosylated residues in proteins is rather tedious. In a previous paper [Biochemistry 42 (2003) 8342], it was reported that the complete analysis of all constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample in three different steps of gas chromatography/mass spectrometry of heptafluorobutyrate derivatives. It was observed that during the acid-catalyzed methanolysis step used for liberation of monosaccharide from classical O- and N-glycans, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively. These derivatives were stable to acid hydrolysis used for the liberation of amino acids. Since monosaccharide derivatives were also stabilized as heptafluorobutyrate derivatives of O-methyl-glycosides, it was suggested that C-mannosides of Trp residues could quantitatively be recovered. Based on the analyses of standard compounds, peptides and RNase 2 from human urine, we report that C (2)-mannosylated Trp could be quantitatively recovered and identified during the step of amino acid analysis. Analyses of different samples indicated that this type of glycosylation is absent in bacteria and yeasts.

Derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis

Carbohydrates, either alone or as constituents of glycoproteins, proteoglycans and glycolipids, are mediators of several cellular events and (patho)physiological processes. Progress in the “glycome” project is closely related to the analytical tools used to define carbohydrate structure and correlate structure with function. Chromatography, electrophoresis and mass spectrometry are the indispensable analytical tools of the on-going research. Carbohydrate derivatization is required for most of these analytical procedures. This review article gives an overview of derivatization methods of carbohydrates for their liquid chromatographic and electrophoretic separation, as well as the mass spectrometric characterization. Pre-column and on-capillary derivatization methods are presented with special emphasis on the derivatization of large carbohydrates.