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Vanderbilt University Mass Spectrometry Core Services

Vanderbilt University Mass Spectrometry Core Services. David L. Hachey, PhD Director of Mass Spectrometry Professor of Pharmacology, Biochemistry, Chemistry. … An Overview.

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Vanderbilt University Mass Spectrometry Core Services

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  1. Vanderbilt University Mass Spectrometry Core Services David L. Hachey, PhD Director of Mass Spectrometry Professor of Pharmacology, Biochemistry, Chemistry

  2. …An Overview • Mass Spectrometry Research Center – A comprehensive, university-wide research facility dedicated to the development of advanced mass spectrometric techniques for the study of complex biological systems. • Research Core – Development of advanced mass spectrometric techniques for the study of complex biological systems. • The Metabolomics & Mass Spectrometry Core – An open-access service facility for qualitative and quantitative analysis of low molecular weight (<2,500 Da) biomolecules. • The Proteomics Core – A specialized service facility for the identification and characterization of proteins and protein complexes. • Tissue Protein Profiling Core – A specialized service facility for profiling of proteins in tissues and biological fluids.

  3. Mass Spectrometry ProteomicsDan Liebler David Friedman Amy Hamm MS CoreDavid Hachey Wade Calcutt Research Richard Caprioli Pierre Chaurand Shannon Cornet Biomarkers Lisa Zimmerman Michelle Reyzer Caprioli Liebler Hachey

  4. J Compton MS Engineer G G Bowersock Bowersock Network Engineer Network Engineer RM Caprioli RM Caprioli M Casey M Casey Admin Officer Admin Officer Director, MSRC Director, MSRC Research Bioanalytical Proteomics Tissue Profiling Maintenance P Chaurand DC Liebler L Zimmerman L Zimmerman DL Hachey Res Assist. Prof Director Director Assoc Co-Director Assoc Co Director DS Cornett DB Friedman DB Friedman Res Instructor M Reyzer MW Calcutt Assoc Co Assoc Co - - Director Director Asst. Director Assoc Co-Director J Mobley A Ham A Ham Res Instructor Research Assistants Research Assistants G Wernecke Assoc Co Assoc Co - - Director Director Software Systems Research Assistants Research Assistants Postdoctoral Fellows Visiting Scholars Graduate Students

  5. …Mass Spectrometry Core • The role of the Metabolomics & MS Core is to provide instrumentation and analytical expertise to the Vanderbilt research community via an open-access instrument facility. • The facility is used each year by about 175 investigators each year in 75 research groups at the University. Overall core instrument use is about 11,000 - 12,000 instrument-hours/year. • The facility is operated as an open-access facility in which users are expected to prepare samples in their labs, then bring them to the Core for analysis. • In order to facilitate research training, the Core sponsors an active MS training program to teach new users how to design experiments, do the sample preparation, operate the instrument and interpret the results. • Core personnel assist users with development of analytical methods when that is the most expedient approach to solving a tough analytical problem.

  6. MS Core Participating Research Programs • Research Center for Pharmacology and Drug Toxicology (PI: JD Morrow) • Center in Molecular Toxicology (PI: FP Guengerich) • General Clinical Research Center (PI: D Robertson) • Vanderbilt Cancer Center (PI: RN DuBois) • Liver-lung Interactions in Lung Inflammation (PI: B Christman) • Molecular and Cellular Basis for Digestive Diseases (PI: DB Polk, R Peek) • Breast Cancer SPORE (PI: CL Arteaga) • Phase I Clinical Trials Center (PI: M Rothenberg) • Lipid MAPS Research Consortium (PI: A Brown) • Glutathione Transferases – Structure & Function (PI: R Armstrong)

  7. …Mass Spectrometry Core Personnel • David L. Hachey, PhD (Core Director) • M. Wade Calcutt, PhD • M. Lisa Manier, M.S. • Betty Fox • Dawn B. Overstreet, B.S. • Lamar Dixon

  8. …Core Operations - Duties of the Director • to establish policies and practices for the equitable use of the facility by all Vanderbilt researchers • to provide regular reports on the use of the facility by Center investigators • to assist in the preparation and submission of grants • to develop an accounting and management system for the facility • to develop a training program on the practical aspects of mass spectrometry • to establish quality control procedures for the use and maintenance of facility instruments • to establish a library of core analytical techniques for general use • to assist in developing new analytical methods

  9. …Core Operations - Techniques & Services • Structural Analysis of Biomolecules • Identification of post translational modifications of proteins and DNA by activated xenobiotic agents • Molecular weight of proteins and peptides by MALDI/TOF • Sequencing and identification of proteins and peptides • Identification of lipid oxidation products • Quantitative Analysis of Drugs in Physiologic Fluids • Quantitative Assessment of DNA oxidative damage • Develop Novel Analytical Methods • Multistage tandem MS techniques for structural analysis • Ultra-trace analysis of biomolecules (<femtomole level) • Analysis of complex mixtures by high-resolution capillary LC/MS. • Train Users in the Art of Mass Spectrometry

  10. Lauren Beihoffer Betty Fox Kerry Fillgrove July, 2004

  11. …Core Operations - Training Program • Instrument Operation • GC/MS • LC/MS • MALDI • Sample Preparation Techniques • GC/MS derivatization chemistry, capillary GC • LC/MS methods optimization • MALDI sample preparation • Data Analysis • Spectrum interpretation • Statistical analysis of quantitative data • QC/QA methods development

  12. …Instrumentation & Facilities GC/MS Hewlett-Packard (Agilent) - 5989A ThermoElectron (Finnigan) - DSQ Tandem LC/MS ThermoElectron (Finnigan) - TSQ 7000 " - TSQ Quantum (x2) " - TSQ Quantum Ultra " - LTQ Linear ion trap " - LCQ Deca XP MALDI/TOF Applied Biosystems - Voyager Elite " - Voyager STR Laboratory Facilities Open-access Instrument Lab - 2,000 sq. ft. Sample Processing Lab - 575 sq. ft. Data Analysis Workstation Lab - 96 sq. ft.

  13. …Core Operations - Facility Management • Instrument Scheduling • FIFO queue based on a signup sheet • Priority: maintenance, routine samples, training, development • Service Fees • GC/MS: $50/hr • LC/MS & MALDI: $60/hr • Core Surcharge: $20/hr • Methods development: $30/hr • Weekend rate TSQ-7000: $30/hr • Training: no charge • Quarterly Use Reports • PPG and Center directors • Individual PI’s

  14. Proteomics Core

  15. Proteomics Laboratory Services • Separation of proteins by 2D SDS-PAGE • 2D Difference Gel Electrophoresis (2D-DIGE) • Simple Protein Identification • Shotgun Proteome Analysis by Multidimensional LC-MS-MS • Identification of Protein Post-Translational Modifications or Protein Adducts • Quantitative Analyses of Differential Protein Expression and Modification

  16. 0.1 0.05 Standardized Log Abundance 0 -0.05 -0.1 -0.15 0 h 0 h 0 h 8 h 8 h 8 h 24 h 24 h 24 h 40 h 40 h 40 h 2D DIGE-MS samples are co-resolved Quantitative inter-comparison of multiple samples (in replicate) MALDI-TOF MS and TOF/TOF MS/MS on targeted proteins

  17. …Proteomics Core LC/MS/MS

  18. Tissue Imaging and Biofluids Core Personnel L to R: Julie Coleman, Michele Reyzer,PhD;, Olivia Faison, Erin Seeley,PhD, and Lisa Zimmerman, PhD.

  19. The Tissue/Biofluids Core Facility provides: Direct proteome profiling and protein imaging of intact tissue by MALDI-MS. Tissue Profiling typically involves many samples of several types, and the goal is often to discover patterns in the protein profiles of the samples that can classify the samples based on biological state. (e.g., tumor vs. normal) and that can predict biological outcomes (e.g., the prognosis of a patient). Tissue Imagingis usually performed on a small number of samples where the goal is to obtain a relatively high resolution image showing the distribution of various proteins in the tissue section. Proteome profiling of serum, plasma and other biofluids by MALDI-MS generates spectral patterns in the protein profiles as a tool used to differentiate between disease states. Multidimensional Analysis (LC-MS-MS) of Biofluids comprehensive analysis of fluids providing inventory of protein identified in samples.

  20. Example of data generated using Tissue/Biofluids Core Facility m/z 10,165 m/z 12,135 High-dose infection (abscesses present) Low-dose infection (abscesses absent) Uninfected (abscesses absent) MS images of mouse kidneys infected with S. aureus. The signal at m/z 10,165, tentatively identified as calgranulin A, is present in the kidney abscesses, but is absent in healthy tissue. The signal at m/z 12,135 appears down-regulated in infected tissue and is absent in abscessed tissue.

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