Kenneth G. Standing

A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance

A matrix-assisted laser desorption/ionization (MALDI) source has been coupled to a tandem quadrupole/time-of-flight (QqTOF) mass spectrometer by means of a collisional damping interface. Mass resolving power of about 10,000 (FWHM) and accuracy in the range of 10 ppm are observed in both single-MS mode and MS/MS mode. Sub-femtomole sensitivity is obtained in single-MS mode, and a few femtomoles in MS/MS mode. Both peptide mass mapping and collision-induced dissociation (CID) analysis of tryptic peptides can be performed from the same MALDI target. Rapid spectral acquisition (a few seconds per spectrum) can be achieved in both modes, so high throughput protein identification is possible. Some information about fragmentation patterns was obtained from a study of the CID spectra of singly charged peptides from a tryptic digest of E. coli citrate synthase. Reasonably successful automatic sequence prediction (>90%) is possible from the CID spectra of singly charged peptides using the SCIEX Predict Sequence routine. Ion production at pressures near 1 Torr (rather than in vacuum) is found to give reduced metastable fragmentation, particularly for higher mass molecular ions. Copyright 2000 John Wiley & Sons, Ltd.

An Improved Model for Prediction of Retention Times of Tryptic Peptides in Ion Pair Reversed-phase HPLC Its Application to Protein Peptide Mapping by Off-Line HPLC-MALDI MS

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-Å pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.

Collisional damping interface for an electrospray ionization time-of-flight mass spectrometer

A new interface between atmosphere and high vacuum has been developed for orthogonal injection of electrosprayed ions into a time-of-flight mass spectrometer. A small rf quadrupole operating at 100 mtorr (1.33 × 10−4 bar) is its key element. Ions enter the quadrupole with velocities acquired in the free expansion/declustering process. As they pass through the quadrupole their motion is constrained by the rf field. Meanwhile, they lose energy by collisions with the gas molecules. The time delays of ions passing through the quadrupole have been measured in order to determine the average velocities of the ions and the factors determining this value. In addition, a simple computational model based on a Monte Carlo approach has been developed to simulate the ion motion; it shows a considerable decrease in both transverse and axial ion velocity components. As the result of collisional damping the interface provides a dramatic improvement in the overall quality of the ion beam transported into the mass spectrometer. Both resolution and sensitivity of the time-of-flight instrument are improved and mass-to-charge ratio discrimination is greatly reduced.

ORTHOGONAL INJECTION OF MATRIX-ASSISTED LASER DESORPTION/IONIZATION IONS INTO A TIME-OF-FLIGHT SPECTROMETER THROUGH A COLLISIONAL DAMPING INTERFACE

Ions are produced from a conventional matrix-assisted laser desorption/ionization (MALDI) target by irradiation with a nitrogen laser pulsed at 20 Hz. After being cooled by collisions in an RF-quadrupole ion guide, the ions enter an orthogonal-injection TOF mass spectrometer, already used for electrospray. The collisional cooling spreads the ions out along the axis of the quadrupole to produce a quasi-continuous beam, which is then pulsed into the mass spectrometer at a repetition rate of about 4 kHz. Approximately five ions enter the mass spectrometer with each injection pulse, and these are detected using single-ion counting and registered in a TDC with 0.5 ns resolution. The performance of the instrument is similar to that obtained with an ESI source. A uniform mass resolution of about 5000 (full width at half maximum definition) is routinely obtained for molecular weights up to about 6000 Da, with mass accuracy around 30 ppm. The sensitivity for peptides is in the low femtomole range. The mass range is currently limited by the low energy (5 keV) of the ions at the detector, although ions of cytochrome C (12 359 Da) have been detected. The performance of the instrument for peptides is competitive with delayed-extraction MALDI in the usual axial geometry, but with the advantage of mass-independent focusing conditions, and a simple two-point calibration procedure. However, the most important advantages result from the nearly complete decoupling of the ion production from the mass measurement. In the usual MALDI experiment the instrument must be carefully adjusted for optimum performance, and the optimum parameters depend on the matrix and the method of sample preparation. As a result of the decoupling, the performance of the instrument is independent of source conditions. This allows much greater flexibility to experiment with different matrices, different substrates (including insulating substrates), and different laser wavelengths, pulse widths and fluences. Because of the decoupling, the design also allows convenient use of both ESI and MALDI sources (and possibly others) on the same spectrometer.

Mass Spectrometric Characterization of Proteins from the SARS Virus A Preliminary Report

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a ∼46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted ∼139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS.

A secondary ion time-of-flight mass spectrometer with an ion mirror

Abstract A new time-of-flight mass spectrometer has recently been constructed at the University of Manitoba (Manitoba TOF II). It incorporates a single-stage ion mirror that enables a resolving power m /Δ m FWHM = 10000 to be obtained, an improvement by a factor ∼ 3 over a previous linear instrument (Manitoba TOF I). The signal/background ratio is also considerably improved. Two detectors are used. One is situated behind the mirror to measure neutral fragments from unimolecular decay in the first leg of the flight path. The other measures the undissociated parent ions and the charged daughters in the reflected spectrum. This system enables measurement of correlations between the charged and the neutral daughter, thus yielding information about the structure of the parent ion.

Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry.

A mass spectrometer coupling electrospray ionization with time-of-flight mass spectrometry (ESI-TOFMS) has been used to investigate the oligomeric species of Escherichia coli citrate synthase, and to determine the effect of nicotinamide adenine dinucleotide (NADH), an allosteric inhibitor of this enzyme, on the equilibrium between the oligomeric forms. An equilibrium mixture of dimers (M = 95,770 Da) and hexamers (M = 287,310 Da) was found under the conditions used (KA = 6.9 x 10(10) M-2), and NADH was observed to bind selectively to the hexamer (KD = 1.1 microM), shifting the equilibrium to the latter form. The power of ESI-TOFMS to measure ions of very large mass-to-charge ratio (up to m/z approximately 10,000 in this case) is shown to be a valuable tool for obtaining accurate information about compositions of noncovalent complexes and equilibrium constants. The measured constants agree with those determined by conventional means.

Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers.

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer. When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits. Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present. On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.

Matrix-assisted laser desorption/ionization quadrupole Time-of-Flight Mass Spectrometry: An elegant tool for peptidomics

A Matrix‐assisted laser desorption/ionization hybrid quadrupole orthogonal acceleration time‐of‐flight mass spectrometer was employed to acquire neuropeptide mass spectra, directly from neuropeptide secreting tissue deposited on the sample target, in the presence of dihydroxybenzoic acid as matrix. The cockroach corpus cardiacum served as model neuroendocrine tissue. Twelve neuropeptide ion peaks, with mass‐to‐charge ratio values ranging between 800 and 3 000 Da were selected for tandem mass spectrometry. All peptides below 1 600 Da could be fully sequenced; tandem mass spectrometry analysis of the remaining (three) largest peptides resulted in (limited) sequence tags, which, also due to unavailability of an appropriate neuropeptide structure database, did not allow complete structure elucidation.

The synovial proteome: analysis of fibroblast-like synoviocytes

The present studies were initiated to determine the protein expression patterns of fibroblast-like synovial (FLS) cells derived from the synovia of rheumatoid arthritis patients. The cellular proteins were separated by two-dimensional polyacrylamide gel electrophoresis and the in-gel digested proteins were analyzed by matrix-assisted laser desorption ionization mass spectrometry. A total of 368 spots were examined and 254 identifications were made. The studies identified a number of proteins that have been implicated in the normal or pathological FLS function (e.g. uridine diphosphoglucose dehydrogenase, galectin 1 and galectin 3) or that have been characterized as potential autoantigens in rheumatoid arthritis (e.g. BiP, colligin, HC gp-39). A novel uncharacterized protein product of chromosome 19 open reading frame 10 was also detected as an apparently major component of FLS cells. These results demonstrate the utility of high-content proteomic approaches in the analysis of FLS composition.