A mass polarizability molecular beam spectrometer

Journal of Biological Physics - A molecular beam spectrometer using dielectrophoresis is described which should be useful in studies of molecular polarizabilities, dipole moments, rotational energy...     

Application and evaluation of limited mass monitoring with a gas chromatograph mass spectrometer computer system

The technique of scanning a preselected set of ions employing a combined gas chromatography mass spectrometer computer system has been investigated to ascertain the advantages and disadvantages of such a procedure. This technique allows one to determine gas chromatographic retention data with with a high degree of precision and accuracy, in rapid temperature programming operation, due to shortening of the mas spectral scanning interval. Signal-to-noise ratio in ion abundance recordings can be enhanced by increasing the dwell time for as many as 100 ions without lenghtening the scanning interval. The utility of such an approach was demonstrated by analysis of complex mixtures isolated form human urine and cerebrospinal fluid.     

Discriminant models for high-throughput proteomics mass spectrometer data

We use several different multivariate analysis methods to discriminate between diseased and healthy patients using protein mass spectrometer data provided by Duke University. Two problems were presented by the university; one in which the responses (diseased or healthy) of the patients were not known and second, when the responses were known. In the latter case, the data can be used as a 'training' set. We attempted both problems. In particular, we use principle component analysis along with clustering methods to discriminate for the first problem set and partial least squares coupled with logistic and discriminant methods when the responses were known. In addition, we were able to detect regions of interest in the spectrum where there were differences in the protein patterns between healthy and diseased patients. There was considerable effort involved in the preprocessing of the data. We used a binning approach to reduce the number of variables rather than peak heights or peak areas. We performed a square root transformation on the data to help stabilize the variance; this in turn made a significant improvement in clustering results.     

Extended performance using a high field magnet mass spectrometer

The development of a high field magnet for high mass electron impact, chemical ionization, field desorption and fast atom bombardment mass spectrometric studies is described. Its utility is illustrated with examples from structural studies of vitamin B12 biosynthetic intermediates, oligosaccharides, glycopeptides and the bleomycin antibiotics. The technique has also greatly assisted sequence studies of protein derived peptides.     

Applications and performance of a MALDI-ToF mass spectrometer with quadratic field reflectron technology

A new matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-ToF MS), developed specifically for the identification and characterization of proteins and peptides in proteomic investigations, is described. The mass spectrometer which can be integrated with the 2-D gel electrophoresis workflow is a bench-top instrument, enabling rapid, reliable and unattended protein identification in low-, as well as high-throughput proteomics applications. To obtain precise information on peptide sequences, the instrument utilizes a timed ion gate and a unique quadratic field reflectron (Z2 technology), allowing single-run, post-source decay (PSD) of selected peptides. In this study, the performance of the instrument in reflectron, PSD and linear mode, respectively, was investigated. The results showed that the limit of detection for a single peptide in reflectron mode was 125 amol with a signal to noise ratio exceeding 20. Average mass resolution for peptides larger than 2000 u was around 13,000 full width, half maximum (FWHM). The limit for protein identification during peptide mass fingerprinting (PMF) was 500 amol with a sequence coverage of 18%. Mass error during PMF analysis was less than 15 ppm for 17 out of 25 (68%) identified peptides. In PSD mode, a complete series of y-ions of a CAF-derivatized peptide could be obtained from 3.75 fmol of material. The average mass error of PSD-generated fragments was less than 0.14 u. Finally, in linear mode, intact proteins with molecular masses greater than 300,000 u were detected with mass errors below 0.2%.     

Screening for EphB signaling effectors using SILAC with a linear ion trap-orbitrap mass spectrometer

Erythropoietin-producing hepatocellular carcinoma (Eph) receptors play important roles in development, neural plasticity, and cancer. We used an Orbitrap mass spectrometer and stable isotope labeling by amino acids in cell culture (SILAC) to identify and quantify 204 proteins with significantly changed abundance in antiphosphotyrosine immunoprecipitates after ephrinB1-Fc stimulation. More than half of all known effectors downstream of EphB receptors were identified in this study, as well as numerous novel candidates for EphB signaling.     

Temporarily immobilized microorganisms: Rapid measurements using a mass spectrometer

Summary Microorganisms were temporarily and reversibly immobilized by entrapment within a small volume. A membrane interface to a mass spectrometer, located downstream from the immobilized microorganisms, allowed direct and continuous measurement of dissolved volatile metabolites.     

Low energy peptide fragmentations in an ESI-Q-Tof type mass spectrometer

Efficient peptide sequencing relies on both high quality MS/MS data acquisition and exhaustive knowledge of gas-phase dissociation mechanisms. We report our contribution to the elaboration of more comprehensive fragmentation models required for efficient automated MS/MS spectra interpretation. Following a statistical approach, various peptides (296 sequences of variable compositions and lengths) were prepared and subjected to low-energy collision-induced dissociations (CID) in an electrospray hybrid instrument (ESI-Q-q-Tof type mass spectrometer) that has retained relatively limited attention so far. Besides, our studies were focused on low molecular weight singly charged peptides that often failed to be identified by sequencing algorithms. Only half of the studied compounds showed charge directed dissociations in accordance with the mobile proton model producing fragment ions directly related to the primary sequence. For the peptides that did not exhibit the expected fragment ion series, alternative dissociation behaviors issued from complex rearrangements were evidenced.     

Biochemical assay by immobilized enzymes and a mass spectrometer.

By counting the volatile molecules produced by an immobilized-enzyme catalyzed reaction which is interfaced to a mass spectrometer via a semi-permeable membrane, a general approach to biochemical measurement and detection is obtained which offers the potential of high sensitivity, specificity and speed. In combination with molecule microscopy, this method should allow, for example, a mapping of suitable enzyme distributions in non-stained and non-fixed tissue slices. Immobilized urease (urea amidohyrdrolase, EC 3.5.1.5) was used to assay urea using CO2 as the volatile product, and alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was used to assay NADH using ethanol as the volatile product.     

18O-labeling quantitative proteomics using an ion trap mass spectrometer

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.     

Microderivatization of 4-[O]hydroxyproline and quantitation with a benchtop mass spectrometer

Accurate estimation of in vivo turnover rates of collagen is complicated by amino acid reutilization. It was previously shown that the ideal, non-recycling tracer was [¹⁸O]hydroxyproline synthesized in vivo. The analytical method for measuring turnover rates with [¹⁸O]hydroxyproline must include analyte quantitation for pool size determination and isotope ratio measurement for determining levels of label incorporation. For ease of use and widest availability, a benchtop gas chromatograph—mass spectrometer in the electron-impact ionization mode was chosen. Here we present a versatile procedure for hydroxyproline derivatization that is well suited for routine, large-scale determination of analyte concentrations and relative levels of ¹⁸O incorporation.     

Exercise with and without gravitational gradient: evaluation with the new random access mass spectrometer (RAMS)

Gravity adds about 40-50 mmHg perfusion pressure to the arterial supply of the quadriceps muscles in the upright posture. This could have important implications in supply of blood flow during exercise. Recently, we have shown that when subjects exercise in the supine posture the rate of increase in VO2 is considerably slower then when cycling exercise takes place in the upright posture. The most probable explanation for this slower adaptation was the altered perfusion gradient. Indeed, when the perfusion gradient from heart to legs was restored by placing the lower part of the body of supine subjects in a negative pressure chamber, the rate of increase in VO2 returned to upright values. The hypothesis advanced from these studies was that skeletal muscle blood flow was reduced at the onset of supine exercise. Exercise in the microgravity environment of space should be similar to that in the supine posture. The only experiments conducted in space to this date that have addressed the question of cardiorespiratory adaptation to changing work rates were performed on the German D2 mission using the methodology proposed by Stegemann and colleagues. To conduct these experiments, it is necessary to utilize sensitive breath-by-breath technology. Recently, NASA and the Russian space programs have commissioned a new mass spectrometer based system as part of the GASMAP project. It was the purpose of this study to evaluate the new mass spectrometer under conditions in which the gravitational effects on the cardiorespiratory response were being challenged.     

New algorithm for 15N/14N quantitation with LC-ESI-MS using an LTQ-FT mass spectrometer

A new algorithm (QN) for the (15)N /(14)N quantitation of relative protein abundances in complex proteomic samples is described. QN takes advantage of the high resolution, mass accuracy and throughput of the hybrid mass spectrometer LTQ-FT MS. Peptide quantitation is based on MS peak intensity (measured in the FT MS), while peptide identification is performed in the MS/MS mode (measured in the LTQ linear ion trap). Accuracy of the protein abundance is enhanced by a novel scoring procedure, allowing filtering of less reliable measurements of peptide abundances. The performance of QN is illustrated in the relative quantitative analysis of M. acetivorans C2A cultures grown with carbon monoxide vs methanol as substrate. Roughly 1,000 proteins were quantitated with an average CV of 9% for the protein abundance ratios. QN performs quantitation without manual intervention, does not require high processing power, and generates files compatible with the Guidelines for Proteomic Data Publication.