Biomedical subjects

Bruce A Thomson

Publications and source records attributed to Bruce A Thomson.

10 recordsLinked to original sources

PubMed2006

Infrared multiphoton dissociation in quadrupole time-of-flight mass spectrometry: top-down characterization of proteins.

The first implementation of infrared multiphoton dissociation (IRMPD) for a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer is reported. Ions were trapped in the radio frequency-only quadrupole (q2), which normally serves as a collision cell, and irradiated by a continuous CO2 IR laser. The laser beam was introduced coaxially with the quadrupoles in order to maximize overlap with the ion path. The resolution of the TOF mass analyzer allowed direct charge state determination for fragments smaller than 7 kDa. For larger fragments, the charge state could be assigned using the multiple losses of water, characteristic for IRMPD of proteins. The analytical performance is demonstrated by top-down sequencing of several representative proteins (equine myoglobin, bovine casein, and human insulin and chaperonin 10). Various post-translational modifications such as phosphorylation, acetylation, formation of disulfide bridges, and removal of N-terminal methionine followed by acetylation are detected and characterized. The utility of IRMPD for the analysis of biological samples is demonstrated in a study of a recently identified potential marker for endometrial cancer, chaperonin 10.

Amino Acid Sequence↗

PubMed2006

The potential of differential mobility analysis coupled to MS for the study of very large singly and multiply charged proteins and protein complexes in the gas phase.

As previously demonstrated by the technique of gas-phase electrophoretic mobility molecular analyzer (GEMMA) introduced by Kaufman and colleagues, differential mobility analysis (DMA) of charge-reduced electrospray ions in the gas phase is a useful complement to MS for studying large proteins and their weakly bound complexes. Several limitations of GEMMA, the solutions for which have the potential to greatly improve its performance, are discussed here, including DMA resolution and transmission. A quantitative theory of charge reduction kinetics for dried multiply charged globular proteins at atmospheric pressures is also presented, showing that the charge reduction time must be carefully chosen to maximize a singly charged ion signal, while avoiding survival of contaminating multiply charged species. Because charge reduction limits the range of masses analyzable by MS, we also consider the potential of a parallel-plate DMA coupled in series to an MS for DMA-MS studies without charge reduction.

Biotechnology↗

PubMed2006

A new hybrid electrospray Fourier transform mass spectrometer: design and performance characteristics.

A new hybrid electrospray quadrupole Fourier transform mass spectrometry (FTMS) instrument design is shown and characterized. This instrument involves coupling an electrospray source and mass-resolving quadrupole, ion accumulation, and collision cell linear ion trap system developed by MDS Sciex with a home-built ion guide and ion cyclotron resonance (ICR) cell. The iterative progression of this design is shown. The final design involves a set of hexapole ion guides to transfer the ions from the accumulation/collision trap through the magnetic field gradient and into the cell. These hexapole ion guides are separated by a thin gate valve and two conduction limits to maintain the required <10(-9) mbar vacuum for FTICR. Low-attomole detection limits for a pure peptide are shown, 220 000 resolving power in broadband mode and 820 000 resolving power in narrow-band mode are demonstrated, and mass accuracy in the <2 ppm range is routinely available provided the signal is abundant, cleanly resolved, and internally calibrated. This instrument design provides high experimental flexibility, allowing Q2 CAD, SORI-CAD, IRMPD, and ECD experiments with selected ion accumulation as well as experiments such as nozzle skimmer dissociation. Initial top-down mass spectrometry experiments on a protein is shown using ECD.

Calmodulin↗

PubMed2005

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins.

The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.

Equipment Design↗

PubMed2005

Combined ion-mobility and mass-spectrometry investigations of metallothionein complexes using a tandem mass spectrometer with a segmented second quadrupole.

Rabbit metallothionein (MT) 2A complexes with Cd(II), Zn(II), Ag(I), Cu(I), Hg(II), arsenite, monomethylarsonous acid (MMA), and dimethylarsinous acid (DMA) have been examined using ion-mobility measurements and mass spectrometry in a triple-quadrupole mass spectrometer equipped with a segmented second quadrupole that doubled as an ion-mobility cell [Guo, Y.; Wang, J.; Javahery, G.; Thomson, B. A.; Siu, K. W. M. An Ion-Mobility Spectrometer with Radial Collisional Focusing. Anal. Chem.2005, 77, 266-275]. The metal ions confer conformational rigidity on the MT complexes, which counteracts Coulombic repulsion among protons added as a result of electrospray. Triply and quadruply protonated Cd(7)MT2A have smaller cross-sections than the Cd(7)MT2A structure deduced from published NMR data. For the 6+ ions, the As(6)MT2A complex has a cross-section of 790 A(2); the MMA(10)MT2A complex, 920 A(2); and the DMA(20)MT2A complex, 1220 A(2). This increase in cross-section of the As(III) species, from As(3+) to MMA to DMA, is interpreted as a consequence of decreasing multiple coordination and increasing number of methyl groups.

Animals↗

PubMed2005

Ion mobility spectrometer with radial collisional focusing.

An ion mobility spectrometer that has its mobility cell as a 20-segment quadrupole and functionally the q2 of a triple-quadrupole mass spectrometer has been assembled and tested. The combination of high cell pressure (maximum of 4 Torr of helium) and low axial field (20-160 V per 20.2 cm) results in negligible internal excitation of the ions despite applications of rf and axial fields. The presence of collisional focusing ensures efficient ion transmission and good sensitivity. Collision cross sections of atomic, cluster, peptide, and protein ions were measured and found comparable to literature and calculated cross sections.

Journal Article↗

PubMed2005

Tandem mobility mass spectrometry study of electrosprayed tetraheptyl ammonium bromide clusters.

Multiply charged electrospray ions from concentrated solutions of Heptyl4N+Br- (designated A+B- hereafter) in formamide are analyzed mass spectrometrically (MS) following mobility selection in ambient air in a differential mobility analyzer (DMA). Most of the sharp mobility peaks seen are identified as (AB)(n)A+ clusters, with 0 < or = n < ot = 5. One anomalously abundant and mobile ion is identified as NH4+(AB)4. Six ions in the (AB)n(A+)2 series are also identified, completing and correcting earlier mobility data for singly and doubly charged ions up to masses of almost 9000 Da. The more mobile of two broad humps seen in the mobility spectrum includes m/z values approximately from 2500 up to 12,000 Da. It is formed primarily by multiply charged (AB)n(A+)z clusters with multiple ammonium bromide adducts. Because of overlapping of many peaks of different m/z and charge state z, only a few individual species can be identified by MS alone in this highly congested region. However, the spectral simplification brought about by mobility selection upstream of the MS reveals a series of broad modulations in m/z space, with all ions resolved in the second, third, ...sixth modulation being in charge states z = 2, 3, ...6, respectively. Extrapolation of this trend beyond the sixth wave fixes the ion charge state (in some cases up to z = 15) and mass (beyond m = 175,000 u). This wavy structure had been previously observed and explained in terms of ion evaporation kinetics from volatile drops, though without mass identification. All observations indicate that the clusters are formed as charged residues, but their charge state is fixed by the Iribarne-Thomson ion evaporation mechanism. Consequently, the measured curve of cluster diameter versus z yields the two parameters governing ion evaporation kinetics. Clusters with z > 1 and electrical mobility Z > 0.495 cm2/V/s are metastable and evaporate a singly charged cluster, probably (AB)2A+, between the DMA and the MS. Plotting the electrical mobilities Z of the clusters in the form (z/Z)1/2 versus m(1/3) (both proportional to cluster diameter) collapse the data for all cluster sizes and charge states into one single straight line for Z below 0.495 cm2/V/s. This linear relation reveals a uniform apparent cluster density of 0.935 g/cm3 and an effective hard-sphere diameter of the air molecules of 0.44 nm. An anomalous mobility increase is observed at diameters below 3 nm.

Bromides↗

PubMed2005

Efficiency of nano-electrospray ionization.

The efficiency of nano-electrospray ionization, defined as the flux of ions reaching the detector of a triple-quadrupole mass spectrometer divided by the flux of analyte ions leaving the needle, has been measured in a series of controlled experiments with dodecyltrimethyl ammonium (DDTMA) bromide, myoglobin, Glu- [1]-fibrinopeptide, and gramicidin S. By varying the flow rate from each needle, the optimum efficiency was determined. In general, the efficiency increased as the flow rate decreased. For DDTMA, efficiencies of up to 12% were measured, although efficiencies of approximately 1% were more common. Ion current measurements indicated efficient transfer of ions from the needle through to the detector. Significant needle-to-needle variations in efficiency were encountered and attributed to variations in ion-generation efficiency.

Equipment Design↗

PubMed2004

Collisional cooling of large ions in electrospray mass spectrometry.

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.

Journal Article↗

PubMed2002

MS3 using the collision cell of a tandem mass spectrometer system.

We report the feasibility of multistage fragmentation in combination with a fast background subtraction method, yielding the equivalent of MS3. The first quadrupole selects an ion of interest, and the ion is axially accelerated into Q2 to generate fragment ions. Subsequent stages of mass selection and fragmentation are obtained by quadrupolar resonant excitation within the Q2 collision cell. The fragments are analyzed downstream by either a resolving quadrupole or a time-of-flight (TOF) mass spectrometer, and multistage spectra are obtained by subtraction (MS(n) - MS(n-1)) for n = 3 or 4. We discuss the characterization of this method, including product ion arrival times, fragmentation efficiencies, and ion selectivity. We report accurate TOF mass spectra of background-subtracted MS3 for protonated molecules reserpine (m/z 609), bosentan (m/z 1552), and taxol (m/z 854).

Bosentan↗