A strategy for the determination of enzyme kinetics using electrospray ionization with an ion trap mass spectrometer.

A simple and rapid means of enzyme kinetic analysis was achieved using electrospray ionization mass spectrometry and a one-point normalization factor. The model system used, glutathione S-transferase from porcine liver, is a two-substrate enzyme catalyzing the conjugation of glutathione with a variety of compounds containing an electrophilic center. An internal standard that is structurally similar to the product was added to the reaction quench solution, and a single-point normalization factor was used to determine the product concentration without the need of a calibration curve. Kinetic parameters, such as Km, Vmax and Ki (for thyroxine), obtained by electrospray mass spectrometry agreed with those obtained from traditional UV-vis spectroscopy, and competitive vs noncompetitive inhibition reactions could be delineated via mass spectrometry. These results suggest that our method can be applied to enzymatic processes in which spectrophotometric or spectrofluorometric assays are not feasible or when the relevant substrates do not incorporate chromophores or fluorophores. This new method is competitive with traditional UV assays in that it is facile and it involves very little analysis time.

Evidence of block and randomly sequenced chondroitin polysaccharides: sequential enzymatic digestion and quantification using ion trap tandem mass spectrometry.

A method for determining the sequence type of the disaccharide repeat region of cartilage samples is introduced. The samples are sequentially subjected to selective and nonselective enzymatic digestion, and the isomeric products from each step are quantified using tandem mass spectrometry. The two-step digestion/quantification protocol identifies whether the global makeup of the polymer is "alternating", "random", or "blocked" with respect to the two main components of the cartilage, 4- and 6-sulfated disaccharides. Using this procedure, the sequence type of two biologically isolated chondroitin polysaccharides was identified. The results for chondroitin sulfate A, isolated from bovine trachea, are consistent with the 4- and 6-sulfated disaccharides randomly distributed throughout the repeat region of the polysaccharide. For chondroitin sulfate C, shark cartilage, the 6-sulfated disaccharides are adjacent to each other to a larger extent than one would expect for a randomly distributed polymer, indicating that "blocks" of repeating disaccharides with the same sulfation site are present.

Molecular orbital considerations in probing the stereoselective dissociations of cobalt-coordinated hexosamine monosaccharides.

The mechanisms for the stereoselective dissociation pathways of isomeric [CoIII(diaminopropane)2(hexosamine-2H)]+ complexes are studied by ion trap and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS). The exact masses of product ions were measured in order to determine the composition of each loss, and isotopic labeling experiments were used to determine which atoms were lost during dissociation. MS3 studies were used to probe the structures of the product ions from MS2 experiments. Based on the experimental evidence obtained, mechanisms explaining the dissociations are postulated. In deciphering the mechanisms, careful attention was paid to the molecular orbital alignment of the reacting bonds, and based on the molecular orbitals, transition state conformations were postulated. These transition states suggest how the observed stereoselectivity occurs. In each case, the carbohydrate/metal interaction was crucial in the dissociation processes.

Utilization of MS3 spectra for the multicomponent quantification of diastereomeric N-acetylhexosamines.

A rapid and accurate means of quantifying mixtures of diastereomeric N-acetylhexosamine monosaccharides using MS3 product ions is introduced. The method involves derivatizing the monosaccharides with [Co(DAP)2Cl2]Cl (where DAP is diaminopropane), and subjecting the derivatized products to collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer. Each diastereomer provides unique MS3 product ion abundances. The abundances for the pure monosaccharide standards are used in a system of equations in order to quantify mixtures of these diastereomers. Using the system of equations is quite advantageous, as it is the only mass spectrometric method that has been shown to successfully quantify mixtures of more than two isomers. The utility of the method is demonstrated by successfully quantifying various two and three component mixtures of the diastereomeric monosaccharides. Furthermore, the method is used to quantify the recovery of a single diastereomeric monosaccharide from an acidic resin. Although the multicomponent quantification method described herein is used to quantify mixtures of N-acetylhexosamine diastereomers, it could be applied to any group of isomers, provided distinguishing CID spectra are obtained. This is the first known report of utilizing MS3 product ions for quantification of structural isomeric mixtures.

Detection and quantification of the sulfated disaccharides in chondroitin sulfate by electrospray tandem mass spectrometry.

A new method of identifying and quantifying the disaccharide building blocks of glycosaminoglycans is introduced. The polysaccharides are subjected to an enzymatic digestion that releases the sulfated disaccharides. The disaccharides are then identified using a combination of electrospray ionization mass spectrometry and tandem mass spectrometry. Quantification of the isomeric disaccharides is also achieved by tandem mass spectrometry, using a recently developed methodology which quantifies mixtures of isomers without the use of chromatography or prior separation. Using mass spectrometry to characterize the components of glycosaminoglycans significantly reduces both sample consumption and analysis time of traditional methods.

Combined partial acid hydrolysis and electrospray ionization-mass spectrometry for the structural determination of oligosaccharides.

A general oligosaccharide acid hydrolysis method, amenable to electrospray ionization mass spectrometry (ESI-MS), is described that allows for hydrolysis of glycosidic bonds for both hexose- and N-acetylhexosamine-containing oligosaccharides. The partial acid hydrolysis of oligosaccharides is obtained by using an acid-exchange resin as the acid catalyst. A ladder sequence of the glycan is produced in solution that is directly analyzed by ESI tandem mass spectrometry, employing both ion trap and Fourier transform ion cyclotron resonance mass spectrometers, to provide sequence and linkage information. Unlike traditional acid hydrolysis procedures, there is minimal degradation of monosaccharide residues or deacetylation of N-acetylhexosamines by employing this technique. It is further demonstrated that the stereochemistry of the released monosaccharides and the anomeric configuration within disaccharides is determined by direct derivatization of the hydrolysate with Zn(dien)-Cl2 followed by ESI-MS/MS.

Multicomponent quantification of diastereomeric hexosamine monosaccharides using ion trap tandem mass spectrometry.

A rapid means of stereochemical differentiation and quantification for the hexosamine monosaccharides was achieved using electrospray ionization quadrupole ion trap mass spectrometry. The hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine, were derivatized with [Co(DAP)2Cl2]Cl, and the complex [Co(DAP)2(HexNH2)]Cl was generated. Subjecting this complex to collision-induced dissociation provided a unique product ion spectrum for each of the diastereomeric monosaccharide complexes, thus differentiating the stereoisomers. Furthermore, the stereoisomers were quantified. This was achieved by using the relative abundances of product ions from pure standards and using these values to determine the ratio of isomeric products in a mixture. The utility of this quantification method was demonstrated by successfully determining the composition of two- and three-component mixtures of the hexosamines.

Differentiation of diastereomeric N-acetylhexosamine monosaccharides using ion trap tandem mass spectrometry.

A quadrupole ion trap mass spectrometer equipped with electrospray ionization was used to distinguish three diastereomeric monosaccharides, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine. The saccharides were derivatized to form the metal complex [CoIII(DAP)2HexNAc]Cl3 which, when collisionally activated, produced dramatically different product ion spectra. The product ion spectra generated for the three monosaccharide diastereomers were then used to confirm the stereochemistry of N-acetylhexosamines from a hydrolyzed oligosaccharide. Finally, the origin of each product ion was determined through isotopic labeling studies, and mechanisms were proposed which explain each resulting dissociation.