MASS SPECTROMETRY OF CARBOHYDRATES CARBOHYDRATES: There are four classes of carbohydrates.

They are Monosaccharide ,Disaccharides ,Oligosaccharides ,Polysaccharides MALDI- MS INTRODUCTION Stachyose , a phytochemical tetrasaccharide, appears to have been the first compound examined .The Solid matrices used are nicotinic acid or tryptophan. The laser source used is requency-quadrupled NdYAG laser (266 nm). The major carbohydrate ion produced by use of these matrices was MNa+(cationisation). The ionization mechanisms involved in MALDI are still largely unknown. Ions may be Pre-formed in the solid state Formed in the gas phase by ionmolecule reactions immediately following desorption by the laser. MALDI INSTRUMENTATION LASER SOURCE: Nitrogen lasers that emit at 337 nm (UV range) are universally employed for MALDI analysis. ANALYSER: The first commercial MALDITOF instrument was produced in 1990 (Cottrell, 1992), but resolution was poor (in the range of 2300). Higher resolutions of around 2000 FWHM (full-height at half-maximum) were soon achieved with magnetic sector instruments and were used for work on carbohydrates (Bordoli et al., 1994, 1996). These instruments had to be fitted with an array detector . Instruments fitted with reflectrons were able to provide even higher resolutions, combined with the advantage of high sensitivity. MATRIX USED Matrices for Free Neutral Carbohydrates Substituted Benzoic Acids 3-Aminoquinoline Matrices for Free Acidic Carbohydrates 6-Aza-2-thiothymine Hydroxyacetophenones Matrices for Sulfated Carbohydrates 1 Nitrocarbazole 7-amino-4-methylcoumarin Sinapic acid COMMON MATRIX USED 1.Substituted Benzoic Acids 2,5-DihydroxyBenzoic Acid This is the popular matrix uesd for carbohydrates. It produces MNa+ species as the major ion. This ion is often accompanied by a weaker MK+ ion, and other

species, such as ML+ can be generated by the addition of the appropriate inorganic salt to the matrix.

Preparation of 2,5-DHB Matrix 2,5-DHB typically crystallizes from mixtures of acetonitrile, or ethanol and water as long, needle-shaped crystals that originate at the periphery of the target spot and project towards the center. The central region usually contains an amorphous mixture of sugar, contaminants, and salts. In order to produce a more even film of crystals, the spot may be re-dissolved in dry ethanol and allowed to recrystallize. This technique not only produced a thin, even film of small crystals, but also increased the sensitivity by about an order of magnitude, probably due to more efficient mixing of sample and analyte from the single solvent. SAMPLE PREPARATION Contaminant removal Derivatisation Contaminant removal Compounds other than the analyte, such as salts and buffers, generally have an adverse effect on ion yield and crystal formation, and they should be removed. MALDI analysis of proteins and glycoproteins appears to be less affected by the presence of these contaminants.Carbohydrates appear to be more susceptible than proteins to the effects of salts and other compounds although the presence of small amounts of sodium or other alkali metals is essential for ionization. EFFECTS OF CONTAMINANTS: Metals have recently been found to cause clustering between the matrix and sample, with adverse effects on resolution. They also cause the matrix multimers, frequently seen with 2,5- DHB in particular, in the region of m/z 2001000 Methods to remove contaminants 1. Removal of salts and buffers from glycoproteins is performed by first adsorbing the glycoprotein onto a gold or better, onto an electrosprayed nitrocellulose target, washing with water, and adding the matrix solution. After about two minutes, the target was allowed to dry as normal, and spectra were obtained. 2a. DROP DIALYSIS: (a floating filter disc replaces the dialysis sac).

Carbohydrates may be cleaned satisfactorily by drop dialysis on a membrane with a reasonably low molecular weight cut-off prior to deposition ontothe target;500 Da is a good cut-off size for the membrane for use with N-linked glycans. 1. Cut a small square of a relevant membrane 1 cm on each side and float this on the surface of water contained in a small container (e.g., a petri dish). 2. Add one or more 1-l drops of an aqueous solution of the glycans to the upper surface of the membrane, cover the apparatus to prevent evaporation, and leave 10 to 15 min at room temperature. 3. Remove the glycan solution and apply directly to a MALDI target. 2b. Nafion-117 membrane is used to to purify sugars. The membrane is pretreated by heating at 80C in nitric or hydrochloric acid for two hours to saturate all of the sulfate groups with protons before the membrane was washed with water. The sample was spotted onto the membrane surface, which was floated on water . Advantage: Adsorbs proteins and peptides and was extremely useful for removing these compounds from glycan mixtures. Used, together with mixed ion-exchange chromatography, for cleaning the released glycan mixtures produced by automated hydrazine release. Used to exchange sodium for other alkali metals in order to produce ions other than MNa+ .Instead of hydrogen, the membrane was saturated with an alkali metal salt and floated on a 100mM salt solution. After about one minute, the sample droplet was recovered with a micropipette and was mixed with matrix as normal. The resulting MALDI ions consisted almost entirely of adducts of the salt used. Method 4: Whittal et al. (1995) first deposited a thick layer of 4-HCCA onto the probe. Deposition of serum sample that contained the target carbohydrates derivatized with tetramethylrhodamine and diluted into 50% ethanol/ water. Immersion of probe in water for 45 sec prior to drying. The excess of water was removed by touching the probe against a wiper. The method enabled the sugar to be detected in serum directly, without the need for an extraction step and with a detection sensitivity of hundreds of fmol/mL. Derivatisation: Reasons for derivatisation: Carbohydrates are not ionized as efficiently as compounds such as proteins.They are not effectively transferred to the vapor phase. Why Go for Derivatisation? Reducing sugars contain a single reactive carbonyl group that can be derivatized separately from the many hydroxyl groups. This property has been used by several investigators to improve ionization and to assist desorption. In order to increase sensitivity, the carbohydrate may be derivatized with a reagent that already contains a charged functional group, or with one that can easily be protonated. Types of derivatisation: o Derivatives Formed by Reductive Amination

o Carbonyl Derivatives o Other Derivatives Derivatives Formed by Reductive Amination The sugar, usually in mild acid solution to promote ring opening, is reacted with a large excess of amine (to form a Schiff base) in the presence of a reducing agent (Scheme 2). The product is a secondary amine, and although this amine can accept a proton during MALDI ionisation.

Example Takao et al. (1996) have used 4-aminobenzoic acid 2- (diethylamino)ethyl ester (ABDEAE) to prepare derivatives of maltoheptaose, dextran, and the N-linked glycan, (Man)8(GlcNAc)2, by reductive amination, and they report sensitivity increases of 1000-fold over that of the free glycan. An MH+ ion rather than the more normal MNa+ ion was formed.

Compounds used for derivatisation

Carbonyl derivatives:
Zhao, Kent, & Chait (1997) used substituted-oxime formation for adding a basic peptide residue in its aminooxyacetyl form to the reducing terminus of several neutral Nlinked glycans, and they reported sensitivity increases of between 50- and 1000-fold. 4-HCCA was used as the matrix, compared with 2,5-DHB/HIQ for the underivatized sugars.

Substituted hydrazones were investigated by Naven & Harvey (1996b) in an attempt to avoid the reduction step of reductive amination with its subsequent problems of reagent removal. The reaction with Girard's T reagent (55) was found to be particularly benecial in producing reasonable increases in sensitivity (10-fold) because of its constitutive cationic charge

Other derivatives PMP (1-phenyl-3-methyl-5-pyrazolone) derivative.Under basic condition reducing sugar reacts with PMP to form PMP derivative. Eg:. Maltooligosaccharides N-linked glycans.

An approach to improve the MALDI spectra of sialic-acid-containing carbohydrates is to methylate the carboxylic acid group of the sialic acid to produce a neutral sugar . This methylation can be accomplished by first converting the acid into its sodium salt with an AG-50 ion-exchange resin in its sodium form, and reacting the resulting sialic acid salt with methyl iodide in dry dimethylsulfoxide (DMSO) for two hours. The free acid can also be methylated, but the reaction takes 48 h to complete. Formation of the methyl ester improves the signal in four ways. It converts the entire ion current into the positive mode, thus avoiding the splitting of the signal between positive and negative ionization. It enables the sialic acid substituted glycans to be measured in the same spectrum as the neutral glycans with equivalent ionization efficiency and, thus, quantitative relationship. It prevents salt formation, with the result that only one peak is produced from each compound, It stabilizes the sialic acid and prevents sialic acid loss by fragmentation Disadvantages of other derivatives Destruction of the pyranose form of the reducing-terminal sugar and the inability to reverse most of the above derivatization reactions to regenerate the native glycan. Fragmentation The fragmentation pathways can be classified into two groups;

Glycosidic cleavages that result from the breaking of a bond linking two sugar rings, The glycosidic cleavages provide information mainly on sequence and branching Cross-ring cleavages that involve the breaking of two bonds. the crossring cleavages reveal more details on linkage.

Types of fragmentation Post-source Decay (PSD) Fragmentation In-Source Decay Fragmentation Fragmentation Produced by Collision-induced Decomposition Post-source Decay (PSD) Fragmentation PSD refers to a method of detecting and measuring the masses of fragment ions that are formed from a selected precursor ion. Fragment ions are mainly formed by unimolecular decomposition after the precursor ions are fully accelerated (after they exit the sourcehence post-source decay) .Fragment ions are separated and detected in the reflector. There are two ways to increase the amount of fragmentation: both act to increase the precursor ions internal energy.By Using higher laser intensity and a collision cell. INSOURCE DECAY FRAGMENTATION Ions formed rapidly within the ion source, which are known as in-source decay (ISD). Collision-induced Decomposition Collision-induced dissociation (CID), referred to by some as collisionally activated dissociation (CAD), is a mechanism to fragment molecular ions in the gas phase.The molecular ions are usually accelerated by some electrical potential to high kinetic energy in the vacuum of a mass spectrometer and then allowed to collide with

neutral gas molecules (often helium, nitrogen or argon). In the collision some of the kinetic energy is converted into internal energy which results in bond breakage and the fragmentation of the molecular ion into smaller fragments. These fragment ions can then be analyzed by a mass spectrometer. Sequence determination of -glucan from Ganoderma lucidum : Ganoderma species (a group of medicinal fungi), including Ganoderma lucidum (Reishi in Japan or Ling-Zhi in China), has been used for a long time in China to prevent and treat various human diseases. -Glucans (beta-glucans) are polysaccharides of D-glucose monomers linked by -glycosidic bonds. They occur most commonly as cellulose in plants, the bran of cereal grains, the cell wall of baker's yeast, certain fungi, mushrooms and bacteria. Some forms of beta glucans are useful in human nutrition as texturing agents and as soluble fiber supplements. Sample preparation: The dried fruiting bodies of G. lucidum were ground and extracted with dd-water (double-distilled water) at 100 degC for 12 hr. The suspension was centrifuged (4000 rpm, 1 hr) to remove the insoluble materials, and the water-soluble crude polysaccharide was obtained (11%). The crude polysaccharide (0.5 g) was degraded into a low molecular weight glucan using a chemical method (2M TFA 50 degC, 2h) and then purified through a gel-filtration chromatography using a Sephadex G-15 column with ddwater as the eluent to obtain a glucan fraction (15~20%). The Matrix used are THAP (2,4,6 trihydroxyacetophenone) and 2,5-DHB (2,5dihydroxybenzoic acid). Experiment: The glucans were exposed to ionisation and the G. lucidum glucans were observed as sodiated ions ([Glcn + Na]+) and the corresponding mass spectrum is given in Figure 1. The mass of the singly charged molecular ion were calculated as 162.14n + 22.990 Da and 162.14n + 22.990 + 18.015 (mass of reducing end residue) Da respectively, where n is the number of glucose units. For example, if n = 10, the mass of the corresponding glucan should be 1662.4 Da respectively. The mass region of ion peaks was observed with a peak-to-peak mass difference of 162.1 Da, consistent with the repeating unit of the -(13)-glucan. REFERENCE: MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY OF CARBOHYDRATES David J. Harvey Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, Pg no-351-334.