Electrospray ionization has recently emerged as a powerful technique for producing intact ions in vacuo from large and complex species in solution. To an extent greater than has previously been possible with the more familiar "soft" ionization methods, this technique makes the power and elegance of mass spectrometric analysis applicable to the large and fragile polar molecules that play such vital roles in biological systems. The distinguishing features of electrospray spectra for large molecules are coherent sequences of peaks whose component ions are multiply charged, the ions of each peak differing by one charge from those of adjacent neighbors in the sequence. Spectra have been obtained for biopolymers including oligonucleotides and proteins, the latter having molecular weights up to 130,000, with as yet no evidence of an upper limit.

detector but not by the mass spectrometer. Those compounds account for the four unidentified peaks appearing between 15 and 30 min in the absorbance chromatogram (Figure 3).The utility of reconstructed mass chromatograms is evident in Figure 4. BHT and Irganox 1076 are easily detected in the reconstructed mass chromatogram, but in the total ion current chromatogram, Irganox 1076 is obscured by the peak at scan 557.

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.

Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.