Harold R. Udseth

An accurate mass tag strategy for quantitative and high-throughput proteome measurements

We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide “accurate mass tags” (AMTs) produced by global protein enzymatic digestion. The two‐stage strategy exploits Fourier transform‐ion cyclotron resonance (FT‐ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from “potential mass tags” identified using conventional tandem mass spectrometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FT‐ICR measurements has been shown capable of characterizing peptide mixtures of significantly more than 105 components with mass accuracies of < 1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian proteomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for >60% of the potentially expressed proteins in the organism Deinococcus radiodurans.

Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organisms dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Collisional activation and collision-activated dissociation of large multiply charged polypeptides and proteins produced by electrospray ionization

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 (Mr) 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) (Mr 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 (Mr 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.

Global Analysis of Deinococcus Radiodurans Proteome by Csing Accurate Mass Tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organisms dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry

The extension of mass spectrometry to high molecular weight biopolymers based upon electrospray ionization and the on-line combination with capillary electrophoresis is described. Electrospray ionization produces gas-phase intact multiply charged molecular ions of biomolecules from highly charged liquid droplets by a high electric field. For high molecular weight substances electrospray ionization results in a characteristic bell-shaped distribution of multiply charged ions, with each adjacent major peak in the spectrum differing by one charge. Multiply charged molecular ions of proteins with molecular weights greater than 130,000 have been observed with a quadrupole mass spectrometer of limited mass-to-charge range (m/z 1700). Molecular weights can be readily determined for large proteins with accuracies in the range of +/- 0.01 to 0.05%; at least an order of magnitude further improvement appears feasible with improved techniques and instrumentation. The electrospray ionization method is sensitive, presently requiring samples in the 100 fmol to 10 pmol range for proteins. Initial results combining rapid separations by capillary zone electrophoresis with on-line mass spectrometric detection via the electrospray ionization source are demonstrated for myoglobin and other proteins and polypeptides. The potential for extension of these methods to molecular weights on the order of 10(6) is discussed.

A NOVEL ION FUNNEL FOR FOCUSING IONS AT ELEVATED PRESSURE USING ELECTROSPRAY IONIZATION MASS SPECTROMETRY

The ability to effectively focus and transmit ions from relatively high pressure ion sources is a key factor that affects sensitivity and dynamic range in mass spectrometry. To improve upon the mass spectrometric sensitivity achievable with electrospray ionization sources a novel ion funnel interface has been developed and implemented with a triple quadrupole mass spectrometer. The ion funnel effectively consists of a series of ring electrodes of progressively smaller internal diamter to which RF and DC electric fields are co-applied. The electric fields create a pseudo-potential causing the collisionally damped ions to be more effectively focused and transmitted as a collimated ion beam. The ion funnel concept we describe is supported by results of SIMION simulations, ion current measurements and implementation with a mass spectrometer. Electrospray ionization mass spectra for an initial ion funnel configuration demonstrated over an order of magnitude increase in signal relative to that of the instrument operated in its standard (capillary inlet-skimmer) configuration under similar conditions.

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.

Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry.

A novel microfabricated device for isoelectric focusing (IEF) incorporating an optimized electrospray ionization (ESI) tip was constructed on polycarbonate plates using laser micromachining. The IEF microchip incorporated a separation channel (50 μ × 30 μ × 16 cm), three fluid connectors, and two buffer reservoirs. Electrical potentials used for IEF focusing and electrospray were applied through platinum electrodes placed in the buffer reservoirs, which were isolated from the separation channel by porous membranes. Direct ESI‐mass spectrometry (MS) using electrosprays produced directly from a sharp emitter „tip”︁ on the microchip was evaluated. The results indicated that this design can produce a stable electrospray and that performance was further improved and made more flexible with the assistance of a sheath gas and sheath liquid. Error analysis of the spectral data showed that the standard deviation in signal intensity for an analyte peak was less than ˜ 5% over 3 h. The production of stable electrosprays directly from microchip IEF device represents a step towards easily fabricated microanalytical devices. Microchannel IEF separations of protein mixtures were demonstrated for uncoated polycarbonate microchips. Direct microchannel IEF‐ESI‐MS was demonstrated using the microfabricated chip with an ion‐trap mass spectrometer for characterization of protein mixtures.

Capillary zone electrophoresis and isotachophoresis— mass spectrometry of polypeptides and proteins based upon an electrospray ionization interface

The special capabilities of the capillary electrophoresis electrospray ionization-mass spectrometer interface for the analysis of peptides and proteins with molecular weights extending to in excess of 100,000 are reviewed. The dynamic combinations of both capillary zone electrophoresis and capillary isotachophoresis with electrospray ionization are illustrated for mixtures of peptides and proteins. Myoglobin and cytochrome c detection limits were ca. 100 fmol. The potential extension of these methods for determination of the primary structure (sequence) of polypeptides using tandem mass spectrometry is shown to be facilitated by the high charge state of ions produced by the electrospray interface. The relevance of these results for advances in analytical biochemistry are discussed.

Charge-state reduction with improved signal intensity of oligonucleotides in electrospray ionization mass spectrometry

The shift of charge states of oligonucleotide negative ions formed in electrospray ionization mass spectrometry to higher mass-to-charge ratio has been accomplished by addition of organic acids and bases to the solution to be electrosprayed. The use of acetic acid or formic acid combined with piperidine and imidazole effectively reduced charge states. Signal intensity and stability were enhanced greatly when the infused solution contained a high percentage of acetonitrile. In addition, the cocktail that contained imidazole, piperidine, and acetic acid in 80% acetonitrile not only reduced charge states, but also substantially suppressed Na adduction. Several oligonucleotides that varied in base composition and length were investigated, and studies of mixtures showed a significant reduction in spectral complexity.