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

这个子项目是众多研究子项目之一

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 both a Thermo-Finnigan LTQ linear quadrupole ion trap liquid chromatograph - mass spectrometer (LCMS) and a Thermo-Finnigan LCQ Deca XP ion trap LCMS. Both instruments have electrospray ionization (ESI) as the primary inlet, and the LTQ is operated in nanospray ESI using an inlet constructed in the Core for micro-LC columns made of fused silica that also function as the nanospray tip. This approach 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. As COBRE investigators begin to understand the nature of the sample preparation for proteomic analysis, we are seeing better success in analyzing samples submitted. 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 the hard part. Dr. Matthews and his staff have worked closely with investigators on data interpretation. Proteomics Analyses by Mass Spectrometry: The following investigators submitted samples that have been measured and completed by the Core during the period 4/1/2007  3/31/2008: PI: Gary Ward Professor Microbiology & Molecular Genetics PI: Mariana Matrajt Assistant Professor Microbiology & Molecular Genetics PI: Mercedes Rinc¿n Associate Professor Medicine  Immunobiology Div. PI: Terrence Delaney Associate Professor Plant Biology PI: Jonathan Boyson Assistant Professor Surgery Each of these projects was being performed by graduate students and postdoctoral fellows in the above laboratories. Individual student or postdoctoral names are not listed. The projects by Gary Ward and Mariana Matrajt focus on Toxoplasma gondii. One manuscript has been prepared and submitted by Dr. Matrajt from work performed. The project by Mercedes Rinc¿n focuses on cell signaling via protein phosphorylation in thymocytes for cellular control. This work has resulted in a manuscript that has been accepted by Science. The project of Dr. Delaney involves plant signaling and plant defense against pathogens in Arabidopsis thaliana. Dr. Boyson is looking to confirm identification of a sub-unit of a protein complex he is currently studying in semi-invariant NKT (iNKT) cells. 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). Two-dimensional gel electrophoresis: All equipment for the analysis and isolation of proteins by 2D gel electrophoresis has been assembled, including the BioRad ExQuest Spot Cutter and a BioRad Microrotofor Cell for prefractionating samples in solution. Dr. Ward and Dr. Haraldsen have generated written protocols for the 2D gel methods available in the Core. We have developed extensive experience in the analysis of samples by 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and we are currently evaluating the new Proteominer technology (BioRad) for the enrichment of low abundance proteins prior to 2D gel analysis. During the period 4/1/2007  3/31/2008, the following investigators made use of the 2D gel capabilities of the Core: PI: Gary Ward Professor Microbiology & Molecular Genetics PI: Ralph Budd Professor Medicine  Immunobiology Div. PI: Chris Huston Assistant Professor Medicine  Infectious Diseases PI: Jonathan Boyson Assistant Professor Surgery Each of these projects was performed by graduate students and postdoctoral fellows in the above laboratories; again, individual student or postdoctoral names are not listed. Projects have included analytical 2D gel electrophoresis, 2D-DIGE and preparative isolation of proteins for identification by LCMS.