Brian E. Winger

High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

A novel electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometer based on a 7-T superconducting magnet was developed for high-resolution accurate mass measurements of large biomolecules. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to the trapped ion cell maintained below 10−9 torr. The increased pumping speed attainable with cryopumping (> 105 L/s) allowed brief pressure excursions to above 10−4 torr, with greatly enhanced trapping efficiencies and subsequent short pumpdown times, facilitating high-resolution mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ES1 source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was generally filled to the space charge limit within 100 ms. The use of 10–25 ms ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (Mr 5734) and ubiquitin (Mr 8565, with resolution sufficient to easily resolve the isotopic envelopes and determine the charge states. The microheterogeneity of the glycoprotein ribonuclease B was examined, giving a measured mass of 14,898.74 Da for the most abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximately 1 ppm for the higher glycosylated and various H3PO4 adducted forms of the protein. Time-domain signals lasting in excess of 80 s were obtained for smaller proteins, producing, for example, a mass resolution of more than 700,000 for the 4+ charge state (m/z 1434) of insulin.

Ion/surface collisions at functionalized self-assembled monolayer surfaces

Abstract Collisions of polyatomic projectile ions at surfaces which bear a variety of organic functional groups result in the scattering of ions which have incorporated groups picked up from the surface. The reactions observed include abstraction of atoms and groups such as H , F , CH 3 and C 2 H 3 . Dissociative ion/surface reactions are also observed; in these, the initial adduct fragments by loss of radical species such as H or F , or loss of a stable neutral molecule such as H 2 , HCN or HF. The strength of the CF bond is suggested to be the reason why closed-shell ions, such as the phenyl cation, are observed to form fluorine addition products, whereas the corresponding hydrogen atom abstraction process is not observed. Surface-induced dissociation (SID) also shows a strong dependence on the nature of the surface. At one extreme is the fluorinated surface, which is a particularly “hard” surface, being effective at transferring projectile translational energy into internal energy. Metal carbonyl ions serve as thermometer molecules to measure translational to internal energy transfer, and experiments with Cr(CO) + 6 and W(CO) + 6 allowed the distribution of internal energy deposited in the projectile ion to be measured. Under typical conditions, the average energy transferred was some 12–19% of the laboratory energy of the projectile, with the higher value corresponding to the fluorinated surface and the lower value to the hydrocarbon surface. The amount of energy taken up by the target in a typical case was approximately 60% of the projectile laboratory energy. The fluorinated surface is also the most efficient among those studied in the total yield of scattered ions produced relative to the projectile ion current. The carboxylic acid terminated alkanethiol is the least efficient of the surfaces studied, whereas the deuterated, hydroxyl, nitrile and ester-linked ferrocene show intermediate behavior. These results indicate that the fluorinated surface has special value in the SID experiment.

Observation and Implications of High Mass-to-Charge Ratio Ions from Electrospray Ionization Mass Spectrometry

High mass-to-charge ratio ions (> 4000) from electrospray ionization (ESI) have been observed for several proteins, including bovine cytochrome c (Mr 12,231) and porcine pepsin (Mr 34,584), by using a quadrupole mass spectrometer with an m/z 45,000 range. The ESI mass spectrum for cytochrome c in an aqueous solution gives a charge state distribution that ranges from 12 + to 2 +, with a broad, low-intensity peak in the mass-to-charge ratio region corresponding to the [M + H]+ ion. the negative ion ESI mass spectrum for pepsin in 1% acetic acid solution shows a charge state distribution ranging from 7− to 2−. To observe the [M - H]− ion, harsher desolvation and interface conditions were required. Also observed was the abundant aggregation of the protens with average charge states substantially lower than observed for their monomeric counterparts. The negative ion ESI mass spectrum for cytochrome c in 1–100 mM NH4OAc solutions showed greater relative abundances for the higher mass-to-charge ratio ions than in acuidic solutions, with an [M - H]− ion relative abundance approximately 50% that of the most abundant charge state peak. The observation that protein aggregates are formed with charge states comparable to monomeric species (at fower mass-to-charge ratios) suggests that the high mass-to-charge ratio monomers may be formed by the dissociation of aggregate species. The observation of low charge state and aggregate molecular ions concurrently with highly charged species may serve to support a variation of the charged residue model, originally described by Dole and co-workers (Dole, M., et al. J. Chem. Phys.1968, 49, 2240; Mack, L. L., et al. J. Chem. Phys.1970, 52, 4977) which involves the Coulombically driven formation of either very highly solvated molecular ions or lower ananometer-diameter droplets.

Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]15+ ion being the most intense peak. With the addition of H2O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.

Proton transfer reaction studies of multiply charged proteins in a high mass-to-charge ratio quadrupole mass spectrometer

Proton transfer reactions of multiply charged ions at high mass-to-charge ratios were explored with a low frequency quadrupole mass spectrometer. This instrument enabled a qualitative comparison of proton transfer reaction rates at low charge states for ions generated by electrospray ionization (ESI) from different solution conformations and for disulfide-linked versus disulfide-reduced protein ions. Proton transfer reactions that efficiently reduced the number of charges for ESI-generated ions to approximately the number of arginines in the polypeptide sequence were observed. No significant differences in gas-phase reaction rates were noted between different solution conformers. Differences in reaction rates between “native” and disulfide-reduced proteins were much smaller than those observed below m/z 2000 with lower proton affinity reagents or by using lower reagent concentrations. These smaller differences in reaction rates are thought to reflect the reduced electrostatic contributions from widely spaced charge sites and thus, the reduced sensitivi ty to an ion’s three-dimensional structure or U compactness.

Isotopic beat patterns in Fourier transform ion cyclotron resonance mass spectrometry: implications for high resolution mass measurements of large biopolymers

Abstract Fourier trasform ion cyclotron resonance (FTICR) mass spectrometry time domain signals from multiply charged biopolymer ions exhibit characteristics and predictable beat patterns due to the closely spaced cyclotron frequencies of the various isotopic constituents. Isotope beat frequencies can readily and accurately be predicted from the difference in cyclotron frequencies of neighbouring isotope peaks. The nature of these signals has important implications for high resolution mass analysis, particularly in instances where rapid spectral acquisition is desirable as with on-line analysis of chromatographic/electrophoretic effluents. Due to the pulsed nature of the frequency information in these transients, resolution improvements are effectively realized in a stepwise nature. As will be demonstrated, the application of apodization functions can have deleterious effects on signal-to-note and resolution when beats are present only near the beginning and end of the transient. Additionally, in instances where the length of FTICR data acquisition is critical (such as in conjunction with on-line separations or in the analysis of very high molecular weight species), it is crucial to choose data acquisition parameters based on the predicted behavior of the time domain signal for the efficient and accurate acquisition of mass spectra.

Characterization of Combinatorial Peptide Libraries by Electrospray Ionization Fourier Transform Mass Spectrometry

The advantages of Fourier transform mass spectrometry (FTMS) are precision high mass accuracy measurements and the capability of high resolution, multistage mass spectrometry together with a number of other advanced features. These powerful facilities can be used to rapidly screen complex mixtures without the necessity of chromatographic separations. The example shown here illustrates the use of the high resolving power and accurate mass capabilities of FTMS for the rapid, direct analysis of a complex mixture, which had been ionized by direct infusion electrospray ionization.

The effects of primary ion energy on the extent of fragmentation in desorption ionization mass spectrometry

Abstract The extent of fragmentation of molecules ionized from the solid state by static and dynamic secondary ion mass spectrometry increases as the translational energy of the bombarding Ar+ or Cs+ ion is decreased. This result applies to both positive and negative ions in the compounds examined, which include ammonium, arsonium, phosphonium, pyridinium, and sulfonate salts, as well as two neutral compounds, a polynuclear aromatic hydrocarbon, and an aromatic carboxylic acid. The degree of fragmentation also depends on the nature of the substrate, with copper consistently yielding more fragmentation than does silver, platinum, or gold. Fragmentation can also be increased by sputter deposition of a thin metal coating over the organic compound. Each of these effects is accounted for in terms of enhanced unimolecular dissociation due to increased internal energy deposition in the desorbed organic ions.

Polymer analysis by photons, sprays, and mass spectrometry

Abstract Commercial mass spectrometers have been available for more than 50 years. Mass spectrometry (MS) has been applied widely to organic analysis, and during the last 20 years applications for the characterization of complex biological macromolecules have steadily spiraled upwards. In sharp contrast, today only a handful of polymer laboratories rely on MS. Why? Here we show what modern MS can offer to polymer chemistry and engineering. This review describes MS results on polymers that have been obtained with newer ionization techniques — matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) and by using time-of-flight and Fourier transform mass spectrometers. Examples illustrating structure and molecular mass determination are presented.

New developments in microscale separations and mass spectrometry for biomonitoring: Capillary electrophoresis and electrospray ionization mass spectrometry

In this article, we briefly highlight the use of capillary electrophoresis for sampling, manipulating, and separating extremely small sample sizes. The extraordinary sensitivity that can be obtained by combined capillary electrophoresis-mass spectrometry is then demonstrated using recent results. We briefly describe the ability to detect noncovalently associated complexes (e.g., double-stranded DNA) by electrospray ionization-mass spectrometry, and conclude with recent results that show the potential for using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry for characterization of biomolecules.