The principles, development, and recent application of electrospray ionization-mass spectrometry (ESI-MS) to biological compounds are reviewed. ESI-MS methods now allow determination of accurate molecular weights for proteins extending to over 50,000, and in some cases well over 100,000. Similar capabilities are being developed for oligonucleotides. The instrumentation used for ESI-MS is briefly described and it is shown that, although ionization efficiency appears to be uniformly high, detector sensitivity may be directly correlated with molecular weight. The use of tandem mass spectrometry (e.g., MS/MS) for extending collision-induced dissociation (CID) methods to the structural studies of large molecules is described. For example, effective CID of various albumin species (molecular weight approximately 66,000) can be obtained, far larger than obtainable for singly charged molecular ions. The combination of capillary electrophoresis, in both free solution zone electrophoresis and isotachophoresis formats, as well as microcolumn liquid chromatography with ESI-MS, provides the capability for on-line separation and analysis of subpicomole quantities of proteins. These and other new developments related to ESI-MS are illustrated by a range of examples. Fundamental considerations suggest even more impressive developments may be anticipated related to detection sensitivity and methods for obtaining structural information.

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Collisional activation (CA) and collision-activated dissociation (CAD) of multiply protonated molecular ions produced by electrospray ionization using an atmospheric pressure source are described. A TAGA 6000E triple-quadrupole mass spectrometer, in both unmodified and differentially pumped inlet arrangements, was used to investigate CA and CAD during transfer through the atmosphere-vacuum interface and subsequent CAD in the tandem instrument. Melittin, which has a molecular weight (M r ) of 2846, is efficiently dissociated in the interface at higher nozzle-skimmer voltages, yielding fragmentation that can be assigned to the various charge states. Selection of such product ions formed in the interface for subsequent tandem mass spectrometry allows confirmation of earlier sequence assignments and extends the utility of these methods. Various charge states of larger polypeptides, such as human parathyroid hormone (1-44) (M r 5064), can be efficiently collisionally dissociated in the second (rf-only) quadrupole. However, for molecular ions of this size, the low-energy collisions used for CAD yield only partial sequence information. For large molecules such as horse heart myoglobin (M r 16,951), the effects of nozzle-skimmer bias are explored, and it is shown that higher charge states (at ≤ m/z 1400) can be effectively dissociated in the interface. Initial results for both metastable (unimolecular) and CAD for myoglobin are reported. The potential and limitations of CAD for large biomolecular ions are discussed. The feasibility of fingerprinting for proteins is illustrated by the CAD spectra of cytochrome c from nine species.

A plasma source ion trap (PSIT) mass spectrometer has been modified to incorporate a radiofrequency octopole ion guide/collision cell between the ion source and the mass spectrometer. This modification allows ions sampled from the plasma to undergo reactions prior to mass spectrometric analysis. This capability can obviate the need for chemical or chromatographic separation of the sample, remove mass spectral intereferences and enable formation of analytically useful molecular ions. Distinct reaction chemistries can be utilized in the octopole and/or ion trap. Performance of the instrument is dramatically improved if hydrogen gas is introduced into the octopole; molecular hydrogen reacts with Ar + and certain other plasma ions, greatly reduces their intensities, and cools and focuses the ion beam prior to its injection into the ion trap.

Electrospray ionization generates multiply charged peptide molecular ions which are efficiently dissociated by collision with a neutral gas to provide sequence-specific daughter ions. The scope for application of this phenomenon is illustrated by the nearly complete sequencing of melittin (relative molecular mass (M,) 2846.5) employing a tandem quadruple mass spectrometer having a mass limit of mlz 1400.Modern "desorption" ionization methods, such as field desorption (FD) fast-atom bombardment (FAB) ,' liquid secondary-ionization mass spectrometry (LSIMS),3 laser desorption (LD),4 and californium-252 plasma desorption (PD)' provide generally useful yields of molecular ions but relatively little fragmentation. Electrospray ionization (ESI)"9 shares this characteristic and also produces multiply charged molecular ions for proteins of mass in excess of 130 kDa.' The effect of this multiple charging is to extend the mass range of analysis in proportion to the extent of the multiplicity of such charging. Useful measurements at molecular weights beyond the current range of conventional ionization sources are therefore accessible with an inexpensive quadrupole mass spectrometer.McLafferty has emphasized the importance of obtaining qualitative information by collision-induced decomposition (CID) with tandem mass spectrometry (MS/MS)" in the absence of significant fragmentation, and with increasing molecular size of an unknown. Numerous examples of peptide-sequence analysis by tandem mass spectrometry have been published in the past 10 years" in some cases providing full-sequence information. In this respect, ACTH (18-39) (relative molecular mass (MI) 2464)'' represents the longest single sequence thus determined. Larger oligopeptides and proteins are currently approached by selective enzymatic or chemical cleavage and analysis of the resulting fragments. For example, the complete primary structure of the redox protein, thiroedoxin, isolated from Chromatium uinosum, was determined from FAB-MS/MS sequence determinations of fourteen chromatographically prepared tryptic peptides (2-18 amino acid residues in length) and several peptides from a Staphylococcus aureus (strain V8) protease digest . I 3 Such studies have demonstrated that MWMS provides a uniquely powerful tool for sequencing" where Edman degradation fails (e.g., because of a blocked NH,-terminus) or is insufficient to elucidate post-translational modifications.Currently, the upper molecular weight for obtaining sequence information by MS/MS methods is generally Author to whom correspondence should be addressed.believed to be about 3000 Da." Numerous reasons are cited for the increasing difficulty for higher molecular weights including decreasing molecular-ion currents, the large number of possible fragmentation pathways, and the decreased efficiency of the collisionalactivation process due to the large internal energies required for dissociation. 14, The result is that "useful" fragmentation is sparse and little qualitative information is obtained.We have reported...