Biomedical subjects

H R Udseth

Publications and source records attributed to H R Udseth.

At least 19 recordsLinked to original sources

PubMed2001

Dynamic range expansion applied to mass spectrometry based on data-dependent selective ion ejection in capillary liquid chromatography fourier transform ion cyclotron resonance for enhanced proteome characterization.

The characterization of cellular proteomes is important for understanding biochemical processes ranging from cell differentiation to cancer development. In one highly promising approach, whole protein extracts or fractions are digested (e.g., with trypsin) and injected into a packed capillary column for subsequent separation. The separated peptides are then introduced on-line to an electrospray ionization source of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer for the detection of peptide accurate mass tags that serve as biomarkers for their parent proteins. In this work, we report the use of data-dependent selective external ion ejection in conjunction with FTICR and on-line capillary LC separations for the enhanced characterization of peptide mixtures and a yeast extract proteome. The number of peptides identified in an LC-FTICR analysis of a yeast proteome digest employing data-dependent rf-only dipolar ejection of the most abundant ion species prior to ion accumulation was 40% higher than that detected in a separate LC-FTICR analysis using conventional nonselective ion accumulation.

Chromatography, Liquid↗

PubMed2001

A multicapillary inlet jet disruption electrodynamic ion funnel interface for improved sensitivity using atmospheric pressure ion sources.

A new multicapillary inlet and ion funnel interface for electrospray ionization-mass spectrometry has been developed and demonstrated to achieve higher ion transmission efficiency compared to a single-capillary inlet and ion funnel interface. Even though the distance between the end of the ESI inlet capillary and the exit of the ion funnel (10 cm) is significantly longer than that of the conventional interface (typically a few millimeters), a significant part of the directed inlet gas flow persists into the first stage of pumping and results in an increased gas load to the second chamber. A jet disrupter made of a circular metal disk placed on axis in the ion funnel enhanced the dispersion of the directed gas flow from a multicapillary inlet and was also found to improve the ion transmission. The ion funnel with the jet disrupter demonstrated a 15% improvement in ion transmission (compared to that without the jet disrupter) and simultaneously reduced the pumping speed required for the first or second stage by a factor of 2-3. Compared to the sensitivity with the standard mass spectrometer interface (an API 3000, Sciex, Concord, ON, Canada) in MS/MS operation using an interface equipped with the jet disrupter and ion funnel, a 5.3-10.7-fold enhancement in signal was observed for samples with concentrations of 100-500 pg/microL and 10.2 to 14.1-fold enhancement for concentrations of 10 to 50 pg/microL. The decreased enhancement at higher concentrations is attributed to space charge effects and detector saturation.

Air Pressure↗

PubMed2001

Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

Animals↗

PubMed2001

Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics.

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.

Chromatography, Liquid↗

PubMed2001

Design and performance of an ESI interface for selective external ion accumulation coupled to a Fourier transform ion cyclotron mass spectrometer.

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid "proteome" analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of approximately 10 zmol (approximately 6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests.

Animals↗

PubMed2001

Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry.

The patterns of gene expression, post-translational modifications, protein/biomolecular interactions, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are generally based upon mass spectrometric analysis of in-gel digested two dimensional polyacrylamide gel electrophoresis (2-D PAGE) separated proteins, allowing relatively rapid protein identification compared to conventional approaches. This technique, however, is constrained by the speed of the 2-D PAGE separations, the sensitivity limits intrinsic to staining necessary for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limited ability for accurate quantitative measurements based on differences in spot intensity. We are presently developing alternative approaches for proteomics based upon the combination of fast capillary electrophoresis, or other suitable chromatographic separations, and the high mass accuracy and sensitivity obtainable with unique Fourier transform ion cyclotron resonance (FTICR) mass spectrometers available at our laboratory. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon approaches for global protein digestion and accurate peptide mass analysis. Quantitation of protein/peptide levels are based on using two or more stable-isotope labeled versions of proteomes which are combined to obtain precise quantitation of relative protein abundances. We describe the status of our efforts towards the development of a high-throughput proteomics capability and present initial results for application to several microorganisms and discuss our efforts for extending the developed capability to mammalian proteomes.

Bacterial Proteins↗

PubMed2001

Dynamically assisted gated trapping for Fourier transform ion cyclotron mass spectrometry.

An efficient approach for trapping ions and enhancing signal based on 'adiabatic amplitude reduction' for Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is described and evaluated. This method is a modification to the widely used gated trapping technique in which the trapping potential is raised adiabatically rather than instantaneously (non-adiabatically). Compared with non-adiabatic gated trapping, the final amplitudes of ion axial oscillations and energies are lower in the proposed method. All performance aspects of the FTICR spectrum (e.g., peak intensities, mass resolution, and mass accuracy) improve significantly compared to the conventional gated trapping technique.

Amino Acid Sequence↗

PubMed2001

A dynamic ion cooling technique for FTICR mass spectrometry.

A fast dynamic ion cooling technique based upon the adiabatic invariant phenomenon for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is presented. The method cools ions in the FTICR trap more efficiently, within a few hundred milliseconds without the use of a buffer gas, and results in a substantial signal enhancement. All performance aspects of the FTICR spectrum, e.g., peak intensities, mass resolution, and mass accuracy, improve significantly compared with cooling based on ion-ion interactions. The method may be useful in biological applications of FTICR, such as in proteomic studies involving extended on-line liquid chromatography (LC) separations, in which both the duty cycle and mass accuracy are crucially important.

Algorithms↗

PubMed2001

Controlled ion fragmentation in a 2-D quadrupole ion trap for external ion accumulation in ESI FTICR mass spectrometry.

Undesired fragmentation of electrospray generated ions in an rf multipole traps can be problematic in many applications. Of special interest here is ion dissociation in a 2-D quadrupole ion trap external to a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) used in proteomic studies. In this work, we identified the experimental parameters that determine the efficiency of ion fragmentation. We have found that under the pressure conditions used in this study there is a specific combination of the radial and axial potential well depths that determines the fragmentation threshold. This combination of rf and dc fields appears to be universal for ions of different mass-to-charge ratios, molecular weights, and charge states. Such universality allows the fragmentation efficiency of the trapped ions in the course of capillary liquid chromatography (LC) separation studied to be controlled and can increase the useful duty cycle and dynamic range of a FTICR mass spectrometer equipped with an external rf only 2-D quadrupole ion trap.

Algorithms↗

PubMed2000

Zeptomole-sensitivity electrospray ionization--Fourier transform ion cyclotron resonance mass spectrometry of proteins.

Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the probability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments implemented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed electrodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 30 zmol (approximately 18,000 molecules) for proteins with molecular weights ranging from 8 to 20 kDa.

Cyclotrons↗

PubMed2000

Design and implementation of a new electrodynamic ion funnel.

A new electrodynamic (rf) ion funnel has been developed and evaluated for use in the interface regions (at approximately 1-10 Torr) of atmospheric pressure ion sources (e.g., electrospray ionization (ESI) for mass spectrometry). The ion funnel consists of a ring electrode ion guide with decreasing i.d. and with a superimposed dc potential gradient along the ring stack. The thicknesses of the ring electrodes and the spacings between them were reduced to 0.5 mm from 1.59 mm compared to those used for previous designs. The new ion funnel displays a significant improvement in low-mass transmission (m/z >200) and sensitivity compared to previous designs. The transmission efficiencies for electrosprayed peptides and proteins (ranging in mass from 200 to 17,000 Da) were typically 50-60% of total incoming currents from a heated capillary inlet. The transmitted ion currents were a factor of 30-56 greater than those of the standard interface for peptide samples and a factor of 18-22 greater than those for protein samples. The sensitivity gains realized at the MS detector were somewhat lower, possibly due to space charge effects in the octapole ion beam guide following the ion funnel. The improved ion transmission properties result primarily from the use of reduced spacings between ring electrodes. We also show that the ion funnel can be operated in two different modes, one using low-rf-amplitude scans, allowing fragile noncovalent complexes (as well as generally undesired adducts) to be transmitted, and the other using high-rf-amplitude scans, providing greater collisional activation and more effective adduct removal (or the dissociation of lower m/z species).

Electrons↗

PubMed2000

Charge capacity limitations of radio frequency ion guides in their use for improved ion accumulation and trapping in mass spectrometry.

The use of radio frequency (rf) ion guides as "linear" two-dimensional ion traps and ion guides for ion storage and accumulation, respectively, is becoming increasingly important for realizing improved sensitivity in mass spectrometry. Analytical relationships describing the ion accumulation operation mode of rf ion guides are reported. Comparisons are made between the rf quadrupole ion guide, higher-order rf multipoles and rf stacked ring ion guides, in terms of the charge capacity limitations due to the instability of ions, rf focusing efficiency limits, and effects due to rf ion heating (i.e., collisional activation due to rf oscillations of ions). Analytical relations for the stored charge quantity are derived in the low ion energy approximation, which is shown to be reasonable for the systems considered. The ion density spatial distribution is derived, an exponential form of which proved to provide a good approximation for high-order rf multipoles and stacked ring rf ion guides. The limit on the stored charge dependence upon rf is shown to be directly related to the thermal dissociation thresholds for the ions being studied; the limitation is weaker for higher-order multipoles and stacked ring ion guides. These results suggest that rf quadrupoles provide an optimum configuration when accumulation of a moderate ion density is sufficient (below 10(9) elementary charges/m). Alternatively, accumulation of an appreciable density for more fragile species, such as noncovalent complexes, may be realized using higher-order multipoles and stacked ring ion guides.

Ions↗

PubMed2000

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 micro x 30 micro x 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 approximately 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.

Animals↗

PubMed2000

Radial stratification of ions as a function of mass to charge ratio in collisional cooling radio frequency multipoles used as ion guides or ion traps.

Collisional cooling in radio frequency (RF) ion guides has been used in mass spectrometry as an intermediate step during the transport of ions from high pressure regions of an ion source into high vacuum regions of a mass analyzer. Such collisional cooling devices are also increasingly used as 'linear', two-dimensional (2D) ion traps for ion storage and accumulation to achieve improved sensitivity and dynamic range. We have used the effective potential approach to study m/z dependent distribution of ions in the devices. Relationships obtained for the ideal 2D multipole demonstrate that after cooling the ion cloud forms concentric cylindrical layers, each of them composed of ions having the same m/z ratio; the higher the m/z, the larger is the radial position occupied by the ions. This behavior results from the fact that the effective RF focusing is stronger for ions of lower m/z, pushing these ions closer to the axis. Radial boundaries of the layers are more distinct for multiply charged ions, compared to singly charged ions having the same m/z and charge density. In the case of sufficiently high ion density and low ion kinetic energy, we show that each m/z layer is separated from its nearest neighbor by a radial gap of low ion density. The radial gaps of low ion population between the layers are formed due to the space charge repulsion. Conditions for establishing the m/z stratified structure include sufficiently high charge density and adequate collisional relaxation. These conditions are likely to occur in collisional RF multipoles operated as ion guides or 2D ion traps for external ion accumulation. When linear ion density increases, the maximum ion cloud radius also increases, and outer layers of high m/z ions approach the multipole rods and may be ejected. This 'overfilling' of the multipole capacity results in a strong discrimination against high m/z ions. A relationship is reported for the maximum linear ion density of a multipole that is not overfilled.

Algorithms↗

PubMed2000

Initial implementation of an electrodynamic ion funnel with Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has become a widely used method to study biopolymers. The method, in combination with an electrospray ionization (ESI) source has demonstrated the highest resolution and accuracy yet achieved for characterization of biomolecules and their noncovalent complexes. The most common design for the ESI interface includes a heated capillary inlet followed by a skimmer having a small orifice to limit gas conductance between a higher pressure (1 to 5 torr) source region and the lower pressure ion guide. The ion losses in the capillary-skimmer interface are large (estimated to be more than 90%) and thus reduce achievable sensitivity. In this work, we report on the initial implementation of a newly developed electrodynamic ion funnel in a 3.5 tesla ESI-FTICR mass spectrometer. The initial results show dramatically improved ion transmission as compared to the conventional capillary-skimmer arrangement. An estimated detection limit of 30 zeptomoles (approximately 18,000 molecules) has been achieved for the analysis of the proteins with molecular weights ranging from 8 to 20 kDa.

Cyclotrons↗

PubMed1999

Characterization of an improved electrodynamic ion funnel interface for electrospray ionization mass spectrometry.

An improved electrodynamic ion funnel for ion focusing at high pressure (> 1 Torr) has been developed for a triple quadrupole mass spectrometer and its performance compared with that of an earlier prototype previously reported. The ion funnel consists of a series of ring electrodes of progressively smaller internal diameters to which rf and dc electric potentials are co-applied. The new design utilizes ring electrodes possessing larger internal diameters that taper down to a relatively larger exit aperture. In the 1-10 Torr pressure range, the new design provides significant improvement in low m/z ion transmission. Additionally, the overall ion transmission range is improved by linked scanning of the ion funnel's rf voltage concomitantly with the scanning of the quadrupole mass analyzer. Transmission of a noncovalent complex through the interface demonstrated that excessive ion heating was not problematic. Computer simulations of ion transport support the ion funnel design and help explain the relative performance of both designs. Both ion simulations and experimental results are in accord and indicate close to 100% ion transmission efficiency for electrosprayed biopolymer ions through the interface and into the mass analyzer.

Animals↗

PubMed1998

An ion funnel interface for improved ion focusing and sensitivity using electrospray ionization mass spectrometry.

To improve upon the already impressive sensitivity achievable with electrospray ionization sources, a novel electrohydrodynamic ion funnel interface has been developed and implemented with a triple-quadrupole mass spectrometer. The ion funnel interface effectively consists of a series of ring electrodes of increasingly small internal diameters to which rf and dc electric potentials are coapplied. In the 1-10-Torr pressure range, the electric fields cause the collisionally damped ions to be more effectively focused and transmitted as a collimated ion beam. This paper describes the ion funnel design and presents an evaluation of its performance using a triple-quadrupole mass spectrometer. Ion transmission and m/z discriminating parameters (resulting in both effective low- and high-m/z cutoffs) are presented based upon both ion current measurements and mass spectra. Electrospray ionization mass spectra of selected protein solutions demonstrated well over 1 order of magnitude increase in signal relative to that of the instrument operated in its standard (inlet capillary-skimmer) configuration under similar conditions. The present results suggest that it will be feasible to realize close to 100% ion transmission efficiency for analytically relevant ions through the electrospray ionization interface and into the mass analyzer.

Animals↗

PubMed1998

On-line dual microdialysis with ESI-MS for direct analysis of complex biological samples and microorganism lysates.

A novel dual-microdialysis approach has been developed for fast and efficient fractionation and cleanup for ESI-MS and ESI MS/MS analyses of biological samples. A modified dynamic microdialyzer utilizing two mirror-image serpentine channels, which sandwich a regenerated cellulose membrane of selected molecular weight cutoff, serves as the first stage for the removal of high-molecular-weight components and cellular residue. The second stage employs a hollow microdialysis capillary to remove low-molecular-weight species (e.g., salts) which can degrade or preclude analysis ESI-MS. A protein mixture consisting of 30 microM bovine serum albumin (BSA), 4.0 microM cytochrome c, 2.3 microM ubiquitin, and 9.4 microM bradykinin in 0.5 M NaCl was used to evaluate the performance of this system. Essentially complete removal of both BSA and NaCl was achieved, resulting in high-quality mass spectra containing only the lower molecular weight proteins. After passing through the on-line dual-microdialysis system, a crude bacteria cell lysate yielded clean ESI-mass spectra in approximately 20 min. MS/MS of selected ions demonstrated abundant fragment ions and provided a second-dimension "fingerprint" of the complex cellular fraction. Preliminary application of this technique for direct characterization of microorganism lysates is presented.

Escherichia coli↗