The objective of this grant is to purchase a FT-MS instrument to expand the mass spectrometry capabilities at UAB in three specific areas: (1) use of H-D exchange to probe protein structure and dynamics, (2) to identify protein modifications (permanent and transient), and (3) to analyze the protein and peptide content of tissue interstitial fluids. The high mass resolution offered by FT-MS. even for large protein molecules, will provide important new capabilities for the study of protein folding using H-D exchange. A focus of research at UAB is in the assembly of virus and phage proteins. Although H-D exchange has been used to study the capsid protein of the HLV virus using MALDI-TOF mass spectrometry, the coverage of the protein with this technique is limited. Preliminary data obtained using Fr-MS at the National High Field Magnet Facility at Florida State University have demonstrated the power of the FT-MS in both the identification of the peptides produced by digestion with pepsin and the high reproducibility of the data obtained. A second important use of FT-MS is in sequencing of peptides that have undergone biochemical or chemical modification. Although we have a Q-tof2 instrument that gives high resolution MS-MS data, accurate selection of the parent ion is not possible with a quadrupole filter. The Fr-MS instrument will also provide the ability to examine protein modification at the intact protein level. This will be valuable for the study of enzyme-substrate-product complexes. This approach is particularly useful for enzymes undergoing a Ping-Pong reaction mechanism where the substrate forms a covalent enzyme intermediate. Thirdly, the ability of Fr-MS to obtain high resolution mass spectral data and at high sensitivity is also very valuable for the study of the components of interstitial fluids. This material is what cells are bathed in and contains many signaling molecules (cytokines, paracrine growth factors etc). Because of the tiny amounts available to the investigator, except for the brain, study of interstital fluids has been largely ignored. Describing the local environment of individual cells is essential for understanding diseases and for the design of drugs to combat them. Overall, the FT-MS instrument will sustain the high quality of biomedical research that is a hallmark of UAB.
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