Successful on-line interfacing of capillary electrophoresis (CE) with electrospray (ES) mass spectrometry (MS) has progressed substantially in recent years. Of particular note also is the development which has occurred in combining the more advanced capillary-based electromigration separation techniques, such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CIT), micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC), with ES/MS. The union of these electromigration schemes with MS detection provides a useful and sensitive analytical tool for the separation, quantitation and identification of biological, therapeutic, environmental and other important classes of chemical analytes. By making optimal use of the characteristics inherent with these separation mechanisms, greatly enhanced MS performance may be obtained. The following review summarizes the significant issues and challenges involved with CE/ES/MS analysis as well as results which have recently been obtained.

The use of an ultrasonic nebulizer to assist electrospray ionization mass spectrometry (ESI-MS) has been described and demonstrated with the analysis of a transfer RNA digest by microcolumn LC. The restricted range of mobile-phase compositions amenable to the electrospray process has traditionally placed a severe limitation on the types of LC applications that can be used with ESI-MS. For this reason, an ultrasonic nebulizer configured for LC has been developed that can generate the fine dispersion of liquid required for ESI-MS from any type of mobile phase. In the case presented here, a transfer RNA was enzymatically digested into its substituent nucleosides, which were then analyzed by microcolumn LC. The required mobile-phase gradient (beginning at 5% methanol) falls outside the solvent range that can be used with conventional electrospray. The ultrasonic nebulizer, however, resolves this problem. The fundamental behavior of the four most common nucleosides (cytidine, adenosine, guanosine, uridine) was studied, and conclusions concerning the effects of solution chemistry were drawn. Specifically, signal from the H+ adducts of these species seems to be strongly dependent on the pKa value. Also, effects from several source operating variables were examined. These included capillary exit voltage, drying and focusing gases, and nebulizer frequency. Performance was found to be consistent over a wide range (0-100% methanol) of mobile-phase compositions. The limit of detection for adenosine injected onto a microcolumn was found to be 100 amol. Finally, nucleosides from as little as 150 fmol of RNA (amount prior to digestion) could be detected.

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Pyridoxal 5-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-L-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP ؉ or NAD ؉ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn 2؉. The protein forms tightly bound dimers. Each monomer has an ␣/␤/␣-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn 2؉ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp 247 and Asp 267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn 2؉ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADPbound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.Pyridoxal 5Ј-phosphate, the catalytically active form of vitamin B 6 , is an important cofactor for many enzymes responsible for the metabolic conversions of amino acids. Vitamin B 6 (pyridoxine) and its derivatives are also efficient singlet oxygen quenchers and potent fungal antioxidants (1). Two different pathways for de novo synthesis of pyridoxine are now recognized. One of these, found in all Archaea, eukaryotes, and in some bacteria, uses the singlet oxygen resistance (SOR1(Pdx1)) gene product, a highly conserved enzyme (2). A number of eubacteria, including Escherichia coli, utilize a specific pathway for pyridoxal phosphate synthesis that is distinct and has been characterized for some time (3). Analysis of sequences from several genomes has revealed that organisms may encode either the SOR1 or E. coli-like pyridoxine biosynthesis genes, but not both (4).The pyridoxal 5Ј-phosphate biosynthesis pathway has been well characterized in E. coli, and consists of six enzymatic steps, beginning with erythrose 4-phosphate and deoxyxylulose 5-phosphate (5-7). All of the enzymes in this pathway have been identified. Of the six enzymes, PdxA, 1 erythromate-4-phosphate dehydrogenase (PdxB), and pyridoxine-5Ј-phosphate oxidase are unique to this pathway (5), whereas SerC and GapA also funct...