VERMONT IMMUNOBIOL / INFECTIOUS DISEASES CTR: CORE C: PROTEOMICS ANALYSIS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 newly acquired Thermo-Finnigan LTQ Orbitrap LCMS capable of high resolution 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. A key function of the past year was continued discussion by Dr. Matthews with the individual COBRE investigators and as talks to the COBRE investigators as a group to relate the capabilities of the Core and how the Core could be used to address scientific problems of COBRE investigators. Special emphasis has been in assisting investigators in preparation of samples for proteomic analyses. Personnel is available in the Core and standard protocols are in place to perform digests of proteins to peptides for proteomic analysis by LCMS, but getting samples to that point is the difficult part. The easy analyses are just that, but many of the samples presented come with a complex set of issues that need to be worked through for a successful analysis. Much of the effort is designing how the mass spectrometry experiment should be done to obtain the data that will answer the question posed by the COBRE investigator. Another key aspect of the Core has been to work with investigators in analysis of their results. The proteomics experiments result in death by data overflow, and reducing data to meaningful terms of protein identification or location of specific posttranslational modifications is both difficult and time consuming. Dr. Matthews and his staff have worked closely with investigators on data analysis. Proteomics Analyses by Mass Spectrometry The following investigators submitted samples that have been measured and completed by the Core during the period 4/1/2008  3/31/2009: Investigator Appointment Department PI: Jonathan Boyson Assistant Professor Surgery +Alan Chant postdoctoral fellow PI: Ralph Budd Professor MedImmunobiology Div. +Andreas Koenig postdoctoral fellow PI: Christopher Huston Assist. Prof Medicine  Infectious Disease PI: Mariana Matrajt Assist. Prof Microbiology & Molecular Genetics +Maria Julia Figueras Graduate student PI: Mercedes Rinc¿n Associate Professor MedImmunobiology Div. +Tina Thornton Research Assoc. PI: Gary Ward Professor Microbiology & Molecular Genetics +Qing Tang Graduate student +Aoife Heaslip 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 and their students focus on Toxoplasma gondii. One manuscript has been prepared and submitted by Dr. Matrajt from work performed. The project by Mercedes Rinc¿n and Tina Thornton focused on cell signaling via protein phosphorylation in thymocytes for cellular control. This work has resulted in a publication in 2008 in Science. Jonathan Boyson is looking to confirm identification of a sub-unit of a protein complex he is currently studying in semi-invariant NKT (iNKT) cells, and Chris Huston is looking to continue targeted global proteomic experiments he began prior to coming to UVM. Ralph Budd and Andreas Koenig similarly are hoping to find protein binding partners to caspases using proteomics approaches. 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).