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Collisional cooling of ions in the rf-only multipole guides has become a method of choice for coupling electrospray sources to various mass analyzers. Normally parameters of such ion guides (length, pressure) provide enough thermalization and focusing for ions in a wide mass range. Noncovalent complexes, however, have more compact conformations than denatured biomolecules of similar mass and, therefore may not be transmitted efficiently through standard ion guides, as demonstrated by theoretical analysis, simulations, and experiments. Several methods of improving collisional cooling for large compact ions have been developed on a quadrupole time-of-flight instrument, which include operating the ion guides at higher pressure and trapping ions to increase the cooling time. Improved transmission of heavy ions obtained with those methods is studied in experiments with proteasome 20S, an oligomeric protein noncovalent complex with molecular weight around 692 000, and a few other compounds.

A hybrid quadrupole time-of-flight mass spectrometer featured with ion trapping capabilities was employed for quantitative profiling of total extracts of endogenous phospholipids. Simultaneous acquisition of precursor ion spectra of multiple fragment ions allowed detection of major classes of phospholipids in a single experiment. Relative changes in their concentration were monitored using a mixture of isotopically labeled endogenous lipids as a comprehensive internal standard. Precursor ion scanning spectra were acquired simultaneously for acyl anions of major fatty acids in negative ion mode and identified the fatty acid moieties and their relative position at the glycerol backbone in individual lipid species. Taken together, a combination of multiple precursor ion scans allowed quantitative monitoring of major perturbation in phospholipid composition and elucidating of molecular heterogeneity of individual lipid species.

A new synthetic and analytical tool, called regiochemical tagging, for the complete identification of structure of isomeric components in a combinatorial mixture is presented.

The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer. The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coil. ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms. Capillary - skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms. Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr). ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108 783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein. Capillary - skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer. The masses of the monomer ions produced in this manner are assigned to the aposubunit, [[subunit + Fe - 3H].sup.+], and [[subunit + 2Fe - 6H].sup.+]. Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13 877.0 Da for the [Alpha]-subunit and 13 517.5 Da for the [Beta]-subunit. Under nondenaturing conditions, a multimeric species with a molecular weight of 110 663 Da is measured by ESI-TOF, corresponding to an [[Alpha].sub.4]/[[Beta].sub.4] octamer. Capillary - skimmer dissociation of the [[Alpha].sub.4]/[[Beta].sub.4] oligomer produces ions corresponding to both types of monomers ([Alpha] and [Beta]) and the corresponding heptamers ([[Alpha].sub.3][[Beta].sub.4] and [[Alpha].sub.4][[Beta].sub.3]). In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86 844 Da. Capillary - skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms.

A novel electrospray ionization time-of-flight mass spectrometer with an ion mirror has been developed. The 'orthogonal acceleration' of ions combined with ion storage modulation enables large sensitivity to be obtained with moderate (R > 2000) mass resolution. Application of the transient recording system allows simultaneous registration of ions of all masses, with sensitivity equal to that in the single ion monitoring regime. The potential of this technique is demonstrated by the primary structure determination of cationic peptides from porcine neutrophils and by the study of the pathways of recombinant proinsulin proteolytic processing catalyzed by various trypsin forms.

Orthogonal injection offers a high-efficiency interface for transferring ions from a continuous beam, such as that in electrospray, to the pulsed mode needed for time-of-flight mass spectrometry. Orthogonal injection of matrix-assisted laser desorption ion spectroscopy (MALDI) ions may be used to solve the limitations of MALDI such as peak broadening from the ion energy spread and inaccurate calibration.

Presents information on electrospray mass spectrometry studies being conducted on non-heme iron-containing proteins. Methods used to obtain proteins; Importance of metalloproteins in the class of biomolecules.