This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Proteomics Core Laboratory for the COBRE has several mass spectrometers available to assist COBRE investigators and their students. There are two Thermo-Finnigan LTQ linear quadrupole ion trap liquid chromatograph - mass spectrometers (LCMS) a Thermo-Finnigan LCQ Deca XP ion trap LCMS, and a Thermo-Finnigan LTQ Orbitrap LCMS capable of high accuracy mass measurement. All instruments have electrospray ionization (ESI) as the primary inlet, and the LTQ instruments are operated in nanospray ESI with micro-LC columns made of fused silica that also function as the nanospray tip. Nanospray improves detection limits for peptide analysis into the femtomole range. The Core continued discussion with the individual COBRE investigators to relate the capabilities of the Core and how the Core could be used to address scientific problems of COBRE investigators. Bryan Balliff, Ph.D. (Assistant Professor of Biology) who has extensive training in proteomics during his postdoctoral studies has also become involved in the Core in assisting several COBRE investigators. Dr. Ballif's laboratory is particularly designed for large scale analysis of protein phosphorylation sites. The methods that his laboratory uses routinely are being incorporated into COBRE investigator sample analyses. Personnel are available in the Core and standard protocols are in place to perform digests of proteins to peptides for proteomic analysis by LCMS, but much of the effort needs to occur in designing experiments to obtain the best mass spectrometry data. Much of the effort by Drs. Matthews and Ballif has been interacting with investigators in experiment design and data analysis for protein identification and location of posttranslational modifications. Proteomics Analyses by Mass Spectrometry The following investigators submitted samples that have been measured and completed by the Core during the period 4/1/2009 ?3/31/2010: Investigator Appointment Department PI: Jason Botten Assistant Professor Med-Immunobiology Div. +Joe Klaus Graduate Student +Phil Eisenhauer Technician +Joanne Russo Technician PI: Ralph Budd Professor Med?Immunobiology Div. +Andreas Koenig postdoctoral fellow +Cheryl Collins Technician PI: Christopher Huston Assist. Prof Medicine ?Infectious Disease +Adam Sateriale Graduate Student PI: Mariana Matrajt Assist. Prof Microbiology & Molecular Genetics PI: Cory Teuscher Professor Med?Immunobiology Div. PI: Gary Ward Professor Microbiology & Molecular Genetics +Jackie Leung Graduate student An important part of the Core is working with graduate student and postdoctoral trainees, exposing them to proteomics and involving them in the sample measurement and data analysis components. The projects by Mariana Matrajt and Gary Ward focus on Toxoplasma gondii. One manuscript has been prepared and submitted by Dr. Matrajt from work performed, another from Dr. Ward was published this year in PLOS Pathogens. The project by Chris Huston is looking to continue targeted global proteomic experiments he began prior to coming to UVM. Ralph Budd and Andreas Koenig similarly are seeking to find protein binding partners to caspases and atypical T-cell receptors using proteomics approaches. Jason Botten's projects focus on the identification of host proteins that associate with arenavirus proteins and was recently awarded an NIH R21 award that heavily involved these proteomic analyses. Cory Teuscher has begun work to identify proteins that interact with histamine receptors and was recently awarded an NIH R01 award in which these proteomics experiments were an important aim. A variety of different LCMS techniques have been used in these projects ranging from identification of as many proteins as possible in the organisms being studied and changes in physiologic and metabolic conditions to focused studies of identification of protein modification sites (e.g. protein phosphorylation site identification).

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory Grants (P20)
Project #
2P20RR021905-06
Application #
8360770
Study Section
Special Emphasis Panel (ZRR1-RI-B (01))
Project Start
2011-07-20
Project End
2012-06-30
Budget Start
2011-07-20
Budget End
2012-06-30
Support Year
6
Fiscal Year
2011
Total Cost
$9,556
Indirect Cost
Name
University of Vermont & St Agric College
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
King, Benjamin R; Hershkowitz, Dylan; Eisenhauer, Philip L et al. (2017) A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR). J Virol 91:
King, Benjamin R; Kellner, Samuel; Eisenhauer, Philip L et al. (2017) Visualization of the lymphocytic choriomeningitis mammarenavirus (LCMV) genome reveals the early endosome as a possible site for genome replication and viral particle pre-assembly. J Gen Virol :
Nock, Adam M; Wargo, Matthew J (2016) Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators. J Bacteriol 198:2503-14
Ziegler, Christopher M; Eisenhauer, Philip; Bruce, Emily A et al. (2016) The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles. PLoS Pathog 12:e1005501
Massilamany, Chandirasegaran; Koenig, Andreas; Reddy, Jay et al. (2016) Autoimmunity in picornavirus infections. Curr Opin Virol 16:8-14
Bonney, Elizabeth A; Krebs, Kendall; Saade, George et al. (2016) Differential senescence in feto-maternal tissues during mouse pregnancy. Placenta 43:26-34
Sateriale, Adam; Miller, Peter; Huston, Christopher D (2016) Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells. Infect Immun 84:1045-53
Ziegler, Christopher M; Eisenhauer, Philip; Bruce, Emily A et al. (2016) A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles. J Gen Virol 97:2084-9
Klaus, Joseph P; Botten, Jason (2016) Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment. J Vis Exp :e53682
Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas et al. (2016) Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity. FEBS Lett 590:1042-52

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