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.

An electrospray ionization source has been coupled to a reflection time-of-flight mass spectrometer. By orienting the ion source perpendicular to the field-free drift region, the longitudinal energy spread of the ion packet has been substantially reduced, allowing a mass resolving power of over 1000 to be achieved for both low-mass and high-mass ions of biological interest. In addition, instrument sensitivity allows the routine detection of low-picomole and subpicomole quantities of large multiply charged species such as cytochrome c (MW = 12,360.9). The potential utility of this instrument for conducting rapid screening of chromatographic effluents is discussed in light of its simplicity, rapid scanning speed, and high sensitivity.

The simultaneous resonant low-energy excitation of leucine enkephalin and its fragment ions was demonstrated in a collision cell of the multipole-quadrupole time-of-flight instrument. Using low-amplitude multiple-resonance excitation CID, we were able to show the exclusive sequential fragmentation of N-and C-terminus fragments all the way to the final fragmentsimmonium ions of phenylalanine or tyrosine. In this CID mode the single-channel dissociation of each new generation of fragments followed the lowest energy decomposition pathways observable on the time scale of our experiment. Up to six generations of sequential dissociation were carried out in multiple-resonance CID experiments. The direct qualitative comparison of fragmentation of axial-acceleration versus resonant (radial) CID was performed in the same instrument. In both activation methods, fragmentation patterns suggested complex decomposition mechanisms attributable to dynamic competition between sequential and parallel dissociation channels. R esonant harmonic excitation of ions is the primary method for MS detection and activation of induced dissociation in most ion traps. Simultaneous excitation of ions of different m/z ratios is routinely employed to illuminate dissociation pathways using ion traps. It can also be used in harmonic ion guides in order to selectively eject or dissociate ions on their way to an MS analyzer. This paper reports results of validation of a nontrapping multiple frequency component low-energy resonant collision-induced dissociation (CID) in a linear quadrupole ion guide.Several of the ion activation methods used in modern MS instruments are selective, by nature, and target exclusively the parent ion, supplying no additional energy to its primary fragments. As examples of such methods, the sustained off-resonance irradiation (SORI) [1], or on-resonance CID in ion cyclotron resonance (ICR) cells [2] can be mentioned. Other activation methods, such as infrared multi-photon dissociation (IRMPD), and its variations [3,4], or black body infrared dissociation (BIRD) [5] are nonselective, by nature, and always excite primary fragments along with the parent ion. A vast majority of ion activation methods may excite primary fragments depending on the condition of the experiment. In such methods, modeling of the dissociation process can be quite complex. For example, in surface induced dissociation (SID) experiments with moderate impact energies, the dissociation event most likely comprises activation of only the parent ion at the surface, followed by its recoil and subsequent unimolecular decomposition. In contrast, at high impact energies, more extensive fragmentation can be observed due to on-surface shattering of the precursor and possibly some of its primary fragments [6].Axial CID in ion guides (or in triple-quadrupoles) achieved by accelerating the primary ions into the collision cell operated at an elevated pressure is a potentially nonselective activation technique. If initial collisions of the projectile ion with the backgr...

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A preliminary design and implementation of a novel approach to electrospray-mass spectrometry are described. Based on a time-of-flight mass analysis, the instrument provides several important advantages for on-line mass analysis: 1, simplicity, ease of use and low manufacturing cost; 2, rapid scan speed, yielding quasi-instantaneous full mass scans at repetition rates up to several kHz; 3, soft ionization and accurate mass determination of extremely large analyte molecules; 4, high sensitivity.