Studies of unusual simple molecules by neutralization-reionization mass spectrometry.

Reactive or unstable molecules are key intermediates in many important reactions, but can be difficult to prepare for experimental studies. Species with missing (:CH-OH) or extra (H3) substituents can often be formed conveniently in the gas phase by neutralizing a beam of a more stable ionic counterpart (CH = O+H, H3+). Reionization of the neutral after approximately 10(-6) seconds tests its stability, whereas its unimolecular chemistry can be probed by preparing it with different amounts of internal energy. The resulting neutral products are reionized and mass analyzed. Isomers are then characterized by ion dissociation and a third mass-analysis step. Many unusual molecules have been characterized with this technique, which can also be used to probe complex unimolecular chemistry, such as that of cyclobutadiene and ethylene oxide.

Tandem mass spectrometry.

Coupling mass spectrometers in series provides a new technique that has many advantages for the analysis of specific organic compounds in complex mixtures. Sensitivity to picograms of targeted compounds can be achieved with high specificity and nearly instantaneous response. The targeted compound is selectively ionized, and its characteristic ions are separated from most others of the mixture in the first mass spectrometer. The selected primary ions are then decomposed by collision, and from the resulting products the final mass analyzer selects secondary ions characteristic of the targeted compound. Tandem mass spectrometry can achieve specificities and sensitivities equivalent of those of methods such as radioimmunoassay and gas chromatography/mass spectrometry, while performing analyses in much shorter times.

Trends in analytical instrumentation.

Methods for deriving chemical information from a variety of systems and environments have changed dramatically in the last decade. Unique principles from physics, chemistry, and biology are the basis for sophisticated instruments that incorporate computers for data acquisition, reduction, and interpretation. Such analytical systems have shown orders-of-magnitude improvements in sensitivity, specificity, and speed, yet with greater simplicity and lower price. The increasing importance of analytical instrumentation requires reexamination of its coverage in educational curricula and of the role of the analytical chemist in its further development and application.

Attomole protein characterization by capillary electrophoresis-mass spectrometry.

Electrospray ionization with an ultralow flow rate (30 kilodaltons) at a resolving power of approximately 60,000 for injections of 0.7 x 10(-18) to 3 x 10(-18) mole of 8- to 29-kilodalton proteins with errors of <1 dalton in molecular mass. Using a crude isolate from human blood, a value of 28,780.6 daltons (calculated, 28,780.4 daltons) was measured for carbonic anhydrase, representing 1 percent by weight of the protein in a single red blood cell. Dissociation of molecular ions from 9 x 10(-18) mole of carbonic anhydrase gave nine sequence-specific fragment ions, more data than required for unique retrieval of this enzyme from the protein database.

Two-dimensional mass spectrometry of biomolecules at the subfemtomole level.

Multiple dimensions of unique molecular structure information can now be obtained from proteins and DNA using mass spectrometry. Less than 10(-16) mol of the active major histocompatibility complex signaling peptide in a mixture of thousands can be identified. For large proteins (> 40 kDa), the high resolving power (> 10(5) and 10(-17) mol sensitivity of Fourier-transform mass spectrometry provide exact molecular weight values (+/- 1 or 2 Da) for mixture components, indicating error or modifications compared with the predicted DNA sequence. Selecting a specific molecular species, its two-dimensional spectrum indicates the part of the molecule that is modified; a three-dimensional spectrum of that fragment further isolates the modification site.

Overexpression of recombinant proteins with a C-terminal thiocarboxylate: implications for protein semisynthesis and thiamin biosynthesis.

A facile and rapid method for the production of protein C-terminal thiocarboxylates on DNA-encoded polypeptides is described. This method, which relies on the mechanism of the cleavage reaction of intein-containing fusion proteins, can produce multi-milligram quantities of protein C-terminal thiocarboxylate quickly and inexpensively. The utility of this method for protein semisynthesis and implications for studies on the biosynthesis of thiamin are discussed.

Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry.

The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy. Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli. Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences. For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue. For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide. The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated. For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.

Analytical information from mass spectrometry, past and future.

Two basic reasons are proposed for the tremendous success and future promise of mass spectrometry: (1) the unusually high volume of data obtainable from unusually small samples and (2) the success in converting these data into structural and quantitative information. The ion abundance dimension of mass spectrometric data is remarkable in its pico-to-ttogram sensitivity and >10(6) dynamic range, and the mass scale dimension is uniquely high in the number of resolution increments for larger molecule ionization and high resolution. Additional dimensions of data arise from chromatographic coupling to mass spectrometry and tandem mass spectrometry, as well as from alternative ionization and ion reaction methods. Converting these data into chemical information is equally important. Past progress in these areas has been cyclical; for the immediate future a greater research emphasis is urged to convert data to information through better understanding of the relevant chemistry and better utilization of modern computer methods.

Organic neutralization agents for neutralization-reionization mass spectrometry.

Porter has shown that excited neutrals of specified internal energies can be prepared by neutralization of an ion beam with metal vapors of low ionization potential (IP). For specific problems in neutralization-reionization mass spectrometry, a metal with the desired IP value may not be available, or it may present experimental problems such as a high vaporization temperature, instrument contamination, or detector instability. The use of organic neutralization agents such as tetra-p-anisylethylene (IP = 6.0 eV) can minimize these problems (although cross sections for neutralization with these are a factor of 5 lower than those with metals), and can provide a much wider range of IP values. Their utility is demonstrated in the neutralization of C4H4 (+•) and CH8 (+•) ions to produce C4H4 and C4H8 of selected internal energies. However, for CH4 (+•) neutralization, the CH4 neutrals formed have a much lower internal energy than predicted, indicating that electron transfer from the neutralization agent predominantly produces its ions in excited states.

193 nm Laser photoionization and photodissociation for isomer differentiation in Fourier-transform mass spectrometry.

Energetic (6.4 eV) multiphoton ionization (MPI) or photodissociation, effected interchangeably in a Fourier-transform mass spectrometer, can differentiate isomers that yield similar electron ionization spectra. Selectivity is shown for isomers of C7H8, C7H9N, C7H7F, C8H10, but not of C6H3Cl{3} and C14H10. The contrasting MPI fragmentations and ionization efficiencies, as well as high sensitivities, are of substantial analytical utility. The high ionization efficiency makes possible high resolution MPI spectra, such as 470,000 (FWHH) for the molecular ion of anthracene, from a single laser pulse.

High resolution and tandem fourier-transform mass spectrometry with californium-252 plasma desorption.

For ions formed by plasma desorption (PD) in a Fourier-transform mass spectrometer, high resolution measurements are demonstrated, such as 65,000 (FWHH) for the protonated molecularion of gramicidin S (MW 1140.7). Resolution is substantially improved by delaying measurements until a significant ion concentration has built up in the cell, and by collisionally deactivating the orbital kinetic energy of the ions. This also makes the ions available for subsequent dissociation steps, so that tandem mass spectrometry can be demonstrated for PD ions. With this for larger ions, collisionally activated dissociation (CAD) is effected with > 85% efficiency. The CAD spectra of (M + Na)(+) and of fragment ions from the PD of gramicidin S provide structurally useful information.

Early gas chromatography/mass spectrometry.

Spectroscopy Laboratory, The Dow Chemical Company, Midland, Michigan, USA In December 1955 or thereabouts, the authors coupled a homemade gas chromatograph to a research time-of-flight mass spectrometer constructed by W. C. Wiley, I. H. McLaren, and D. B. Harrington. This unique gas chromatography/mass spectrometry (GC/MS) instrument generated mass spectra at a lo-kHz rate for display on an oscilloscope; eluted gas chromate graphic components, such as methanol, acetone, benzene, toluene, and carbon tetrachloride, could be visually identified immediately from the oscilloscope display. Many years of further research and development in many laboratories worldwide were necessary, however, to make continuous on-line GC/MS the uniquely valuable analytical tool that it is today.

Long-lived metallized tips for nanoliter electrospray mass spectrometry.

Sheathless electrospray at nL/min flow rates combined with Fourier-transform mass spectrometry has made possible high resolving power (>50,000) mass spectra of subattomole samples of >8 kDa proteins separated by capillary electrophoresis (Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science, 1996, 273, 1199-1202). However, for this new method the mechanical stability of the thin (35 to 100 nm) gold film electrodes has limited tip lifetime to 15 to 30 min. A technique for SiO x coating of the gold is described that provides a steady ion current (±10 pA) for 1 to 2 h, even with arcs or interruptions of the electrospray voltage.

Infrared photodissociation of non-covalent adducts of electrosprayed nucleotide ions.

Efficient dissociation of gaseous non-covalent adducts of multiply charged DNA anions can be effected by infrared irradiation to yield minimal dissociation of the DNA molecular ions-far less dissociation than by collisional activation. Examples include removal of adducted impurities from 100-mer DNA anions and removal of all 14 adducted molecules of 1,4-diaminobutane from M(15-) to M(17-) of a 50-mer DNA.

Protonated ethanol and its neutral counterparts.

Collisionally activated dissociation and neutralization-reionization experiments reveal that protonation of ethanol leads to two distinct isomers, the classical ion CH3CH2OH+2 and the proton-bound complex C2H4… H+… OH2. The neutral counterpart of the latter is unstable, whereas that of the former can be produced in a bound state if the CH3CH2OH+2 precursor ion is formed under low ion source pressure conditions and, thus, with higher internal energies. This suggests that there are substantial differences in the geometries of CH3CH2OH(+) 2 and the hypervalent CH3CH2OH2·. This provides only a partial explanation for unusual isotope effects; C2H5OD2·, CH3CD2OD2·, and CD3CH2OD2. are substantially more stable than C2D5OD2 · and C2H5OH2·.

Angle-resolved neutralization-reionization mass spectrometry.

Neutralization -reionization mass spectra of 2-propenal, isomeric butenes, and isomeric n-hexenes have been found to depend significantly on the z-axis scattering angle of the neutralization event. As shown by Cooks for ion dissociations, increasing scattering angles generally favor products of higher activation-energy reactions. For isomeric butenes and n-hexenes, these reactions provide more definitive information for isomeric characterization.

High-resolution ion isolation with the ion cyclotron resonance capacitively coupled open cell.

For ion cyclotron resonance, a capacitively coupled open cell variant with fourfold radial symmetry was constructed and tested for axial excitation-ejection of large ions at high resolution. With a reverse of frequency sweep direction, this cell gave substantial improvements in signal-to-noise ratio due to linearization of the excitation electric field. Single isotopic peaks of ubiquitin (8.6-kDa) and carbonic anhydrase (29-kDa) molecular ions could be isolated by selective stored waveform inverse Fourier transform excitation, which yielded an order of magnitude higher isolation resolving power than previously achieved at high mass-to-charge ratio values.

Electrospray mass spectra from protein electroeluted from sodium dodecylsulfate polyacrylamide gel electrophoresis gels.

Electrospray ionization/tandem mass spectrometry of proteins separated on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels is severely limited by the requirement that the protein be completely separated from the SDS. As shown here, the gaseous noncovalent SDS adducts of protonated proteins thus formed can be dissociated by infrared irradiation. ESI spectra from myoglobin in SDS-containing solutions show molecular ions adducted with up to 15 dodecyl sulfates, but ir irradiation of these ions causes complete dissociation to expel the SDS.

Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules.

Here a fully automated computer algorithm is applied to complex mass spectra of peptides and proteins. This method uses a subtractive peak finding routine to locate possible isotopic clusters in the spectrum, subjecting these to a combination of the previous Fourier transform/Patterson method for primary charge determination and the method for least-squares fitting to a theoretically derived isotopic abundance distribution for m/z determination of the most abundant isotopic peak, and the statistical reliability of this determination. If a predicted protein sequence is available, each such m/z value is checked for assignment as a sequence fragment. A new signal-to-noise calculation procedure has been devised for accurate determination of baseline and noise width for spectra with high peak density. In 2 h, the program identified 824 isotopic clusters representing 581 mass values in the spectrum of a GluC digest of a 191 kDa protein; this is >50% more than the number of mass values found by the extremely tedious operator-applied methodology used previously. The program should be generally applicable to classes of large molecules, including DNA and polymers. Thorough high resolution analysis of spectra by Horn (THRASH) is proposed as the program's verb.

Comparative evaluations of mass spectral data bases.

Recent reports from the National Institute of Science and Technology (NIST) state that its large (53,994) collection of mass spectra is unique in "consisting almost entirely of complete spectra." Our study of the 1989 Registry of Mass Spectral Data of 139,859 different spectra shows that its 53,994 spectra containing the most peaks average 108 peaks per spectrum, 48% larger than the NIST data base. Further, in matching unknown spectra of compounds present in both files, by using criteria yielding 68% reliability, 14% of the possible correct answers were recalled with the NIST data base versus 36% with the Registry.