This proposal seeks funding to purchase a hybrid linear ion trap-orbitrap mass spectrometer that will become part of a shared hydrogen/deuterium exchange mass spectrometry (HDX MS) system dedicated to the study of protein dynamics of large protein complexes and membrane proteins such as G-protein coupled receptors (GPCRs). This shared instrument will support a wide range of research projects originating from ten NIH funded investigators. We present justification for this state-of-the-art mass spectrometer by demonstrating that significant increases to the information content can be obtained from HDX MS experiments performed with an Orbitrap mass analyzer. An instrument resolving power of e 100,000 has been established to be optimal for the baseline resolution of peptide isotope clusters. The current HDX MS platform at Scripps lacks the ability to perform tandem mass spectrometry experiments with high resolution (100,000) and mass accuracy (sub 2 ppm) which will be essential for the complete characterization of complex proteins and large protein complexes. For example, GPCRs are heavily posttranslationally modified;two sites of N-glycosylation are known to contain multiple glycan structures (up to five different sugars at each site), there are multiple sites of phosphorylation, intra molecular disulphide linkages and two sites of protein palmitoylation. Each of these sites of modification requires complete characterization in order to obtain comprehensive HDX kinetics. The complexity of these integral membrane proteins requires a state-of-the-art mass spectrometer such as the LTQ-orbitrap. One specific use of this shared instrument is the study of transcriptional complexes associated with diseases such as diabetes and cancer. Also, as a collaboration of three major users, this instrument will facilitate HDX analysis of GPCRs upon ligand binding. GPCRs represent an important protein family targeted for therapeutic intervention in many diseases. This shared instrument will be installed in the Scripps Florida Mass Spectrometry Laboratory and it will be maintained by a group of very experienced mass spectroscopists, the PI and two Senior Scientists. Several Post Doctoral fellows will be trained or continuing their training on this instrument. The instrument will also be supported by two research technicians. Graduate students in the Kellogg School and attending Florida Atlantic University will also be trained. An appropriate management committee has been put in place to manage instrument time amongst current major users and to facilitate the integration of new users.
This proposal seeks funding to purchase a hybrid linear ion trap-orbitrap mass spectrometer that will become part of a shared hydrogen/deuterium exchange mass spectrometry (HDX MS) system dedicated to the study of protein dynamics of large protein complexes and membrane proteins such as G-protein coupled receptors (GPCRs). One specific use of this shared instrument is the study of transcriptional complexes associated with diseases such as diabetes and cancer and as a collaboration of several major users this instrument will be used to study the dynamics of GPCRs upon ligand binding. This instrument will also provided training for research technicians, post doctoral fellows, and graduate students at the Kellogg School and at Florida Atlantic University.
|Zheng, Jie; Chang, Mi Ra; Stites, Ryan E et al. (2017) HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions. Nat Commun 8:923|
|Francis, Joshua W; Goswami, Devrishi; Novick, Scott J et al. (2017) Nucleotide Binding to ARL2 in the TBCD?ARL2??-Tubulin Complex Drives Conformational Changes in ?-Tubulin. J Mol Biol 429:3696-3716|
|Stechschulte, L A; Czernik, P J; Rotter, Z C et al. (2016) PPARG Post-translational Modifications Regulate Bone Formation and Bone Resorption. EBioMedicine 10:174-84|
|Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose et al. (2015) Integration of G protein ? (G?) signaling by the regulator of G protein signaling 14 (RGS14). J Biol Chem 290:9037-49|
|Marciano, David P; Kuruvilla, Dana S; Boregowda, Siddaraju V et al. (2015) Pharmacological repression of PPAR? promotes osteogenesis. Nat Commun 6:7443|
|Kang, Yanyong; Zhou, X Edward; Gao, Xiang et al. (2015) Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523:561-7|
|Goswami, Devrishi; Tuske, Steve; Pascal, Bruce D et al. (2015) Differential isotopic enrichment to facilitate characterization of asymmetric multimeric proteins using hydrogen/deuterium exchange mass spectrometry. Anal Chem 87:4015-4022|
|Yang, Linlin; Yang, Dehua; de Graaf, Chris et al. (2015) Conformational states of the full-length glucagon receptor. Nat Commun 6:7859|
|Kojetin, Douglas J; Matta-Camacho, Edna; Hughes, Travis S et al. (2015) Structural mechanism for signal transduction in RXR nuclear receptor heterodimers. Nat Commun 6:8013|
|Kirschke, Elaine; Goswami, Devrishi; Southworth, Daniel et al. (2014) Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles. Cell 157:1685-97|
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