Resource for Biomedical and Bio-Organic Mass Spectrometry

Resource for Biomedical and Bio-Organic Mass Spectrometry

Research Emphasis

The focus of this resource is to provide access to state-of-the-art mass spectrometry (MS) instrumentation for researchers through collaborative research and service arrangements, maintain a broad range of MS instrumentation well-suited to studies of biomolecules, and educate students in the MS arts. A unique characteristic of this resource is the existence of one laboratory in the chemistry department and two laboratories in the medical school, one of which is devoted to proteomics.

Current Research

Instrument and method development in tandem quadrupole time-of-flight, ion trap, and Fourier transform MS (FTMS) are aims at the chemistry site. Special emphases are on the development of ionic liquid matrices for matrix-assisted laser desorption ionization (MALDI) and the use of FTMS as the mass analyzer for MALDI. In biophysics, the purpose is to determine structure and properties of peptides and proteins, with emphasis on H/D exchange and OH radical footprinting.

Studies of complex mixtures of lipids and proteins and investigations using stable isotope tracers are aims at the medical school site. A new laboratory for research proteomics was recently made part of the resource. Development of new methods for the analysis of posttranslational modifications and membrane glycoproteins are major goals of the proteomics laboratory. Specific applications are the metabolism of proteins, glucose, and fatty acids and the role of lipids in the biochemistry of insulin-producing cells.

Resource Capabilities

Methods

Staff at the resource are expert in proteomics, complex lipid analysis, biophysical applications of MS (e.g., H/D amide exchange of proteins), and accurate mass measurements. Methods are in place for the analysis of complex mixtures of peptides in proteomics and immunology, complex lipids, and various chemical footprinting methods for proteins (H/D exchange and OH radical foot printing).

Instruments

Equipment at the chemistry site includes two Finnigan capillary liquid chromatography (LC) ion traps (LCQ), an ABI Voyager DE time-of-flight (TOF) mass spectrometer with MALDI, a Micromass quadrupole TOF (QTOF) tandem spectrometer equipped with electrospray ionization and MALDI, a Thermo ion-trap FTMS instrument (LTQ FTMS) with capillary high-performance liquid chromatography (HPLC) for proteomics and protein biophysics, an Ion Spec 7 Tesla MALDI instrument for instrument development and applications, and two tandem magnetic sectors.

Equipment at the medical school site includes MALDI TOF (ABI-DE-STR) and MALDI-TOF/TOF (ABI Proteomics 4700) instruments (of the highest mass resolution for commercial MALDI instruments); Finnigan LCQ DECA and LTQ ion-trap instruments; a Micromass QTOF Micro hybrid QTOF LC/MS/MS system; a Sciex API Q-STAR Pulsar; a Finnigan TSQ 7000 triple quadrupole LC/MS/MS system; a Finnigan TSQ Quantum triple quadrupole LC/MS/MS system with extended m/z range. The LTQ and QSTAR are equipped with one- and two-dimensional automated Eksigent nano-LC systems. A Finnigan SSQ 7000 single-quadrupole with EI/CI and +/- ion capabilities and extended m/z range; two Hewlett-Packard 5970 quadrupole gas chromatography (GC)/mass spectrometers with EI/CI and +/- ion capabilities are available for GC/MS. Special equipment is a Finnigan isotope ratio instrument with online combustion-reduction capabilities for analyses of ¹³C/¹²C, ¹⁵N/¹⁴N, ¹⁸O/¹⁶O, and ²H/¹H.

Software

Staff at the resource are developing software for motif identification for antigenic peptides and for identification of posttranslationally modified peptides in proteomics.

Special Features

Research emphases are H/D exchange and chemical footprinting of proteins to characterize interactions and affinities, structure determination of lipids, isotope ratio MS in metabolism, and trace level isotope dilution quantitation of biomolecules, including intermediatry metabolites, autacoids, and signaling molecules. This is the only NCRR with isotope ratio measurement capability. The chemistry site offers accurate mass measurements of small and midsized molecules and analysis by LC/MS. The proteomics laboratory is available for quantitative analysis of complex mixtures of proteins using two-dimensional difference gel electrophoresis and stable isotope labeling.

Available Resources

Staff members at the resource are eager to collaborate with biomedical researchers in analyzing complex lipids; with immunologists in characterizing complex mixtures of antigenic peptides; with biophysicists in using mass spectrometric methods to determine protein folding, ligand binding, protein/ligand interfaces, protein-protein interactions, and solvent accessibility; and with biomedical researchers in solving difficult problems in proteomics. A major cross-campus focus for the resource is the mitochondrial muscle proteome.

Training Opportunities and Workshops

Scientists are welcome to arrange visits to the resource to conduct, in collaboration with staff, specialized experiments in MS that relate to protein or lipid biochemistry and biophysics. Visiting scientists on brief or extended sabbaticals are also welcome. The resource sponsors, along with the American Chemical Society, a local discussion group in MS, a seminar program in MS in the medical school, and occasional workshops and symposia.

Publications

1. Bao, S., Bohrer, A., Ramanadham, S., Jin, W., Zhang, S., and Turk, J., Effects of stable suppression of group VIA phospholipase A2 expression on phospholipid content and composition, insulin secretion, and proliferation of INS-1 insulinoma cells. Journal of Biological Chemistry 281:187–198, 2006.

2. King, J. B., Gross, J., Lovly, C. M., Rohrs, H., Piwnica-Worms, H., and Townsend, R. R., Accurate mass-driven analysis for the characterization of protein phosphorylation. Study of the human Chk2 protein kinase. Analytical Chemistry 78:2171–2181, 2006.

3. Hambly, D. M. and Gross, M. L., Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale. Journal of the American Society for Mass Spectrometry 16:2057–2063, 2005.