vanderbilt university mass spectrometry core services david l. hachey, phd director of mass...
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Vanderbilt University Mass Spectrometry Core
Services
David L. Hachey, PhDDirector of Mass Spectrometry
Professor of Pharmacology, Biochemistry, Chemistry
…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.
Mass Spectrometry
MS CoreDavid HacheyWade Calcutt
ProteomicsDan Liebler
David Friedman
Amy Hamm
ResearchRichard CaprioliPierre ChaurandShannon Cornet
LieblerCaprioli
Hachey
BiomarkersLisa ZimmermanMichelle Reyzer
RM CaprioliDirector, MSRC
RM CaprioliDirector, MSRC
DL HacheyDirector
M CaseyAdmin Officer
M CaseyAdmin Officer
MW Calcutt
Asst. Director
J ComptonMS Engineer
P ChaurandRes Assist. Prof
J MobleyRes Instructor
PostdoctoralFellows
GraduateStudents
ResearchAssistants
VisitingScholars
DC LieblerDirectorDirector
L ZimmermanAssoc Co
L ZimmermanAssoc Co-Director
Research Bioanalytical Proteomics
ResearchAssistants
DS CornettRes Instructor
A HamAssoc Co-Director
A HamAssoc Co-Director
DB FriedmanAssoc Co-Director
DB FriedmanAssoc Co-Director
G BowersockNetwork Engineer
G BowersockNetwork Engineer
M Reyzer
Research Assistants
Tissue Profiling Maintenance
ResearchAssistants
G Wernecke
Assoc Co-Director
Software Systems
• 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 toprepare 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.
…Mass Spectrometry Core
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)
…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
…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
…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
Lauren Beihoffer Betty Fox Kerry Fillgrove
July, 2004
…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
…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.
…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
Proteomics Core
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
24 h
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Quantitative inter-comparison of
multiple samples (in replicate)
Cy3:
Cy5:
Cy2:
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gel # 1 2 3 4 5 6
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MALDI-TOF MS and TOF/TOF MS/MS on targeted proteins
Cy3 Cy5
image analysis software
image gel
single 2D gel
mix
Cy3 Cy5sample sample
Cy2
Cy2standard
Cy3 Cy5
image analysis software
image gel
single 2D gel
mix
Cy3 Cy5sample sample
Cy3 Cy5
image analysis software
image gel
single 2D gel
mix
Cy3 Cy5sample sample
Cy2
Cy2standard
Cy2
Cy2standard
…Proteomics Core LC/MS/MS
MS and Proteomics Services and Fees
Service Fee
Capillary GC/MS setup and analysis $50 per instrument hour
Tandem LC/MS $60 per instrument hour
MALDI/TOF $60 per instrument hour
Analytical method development & validation $30 per hour
Separation of proteins by 2D SDS-PAGE $140 per gel
2D Difference Gel Electrophoresis (2D-DIGE) $300 – 330 per gel
Excission and processing of 1D or 2D gel spots for tandem MS $50 per spot
Simple Protein Identification by Tandem LC/MS $60 per hour
Shotgun Proteome Analysis by Multidimensional LC-MS-MS $500 per sample
Identification of Post-Translational Modifications by LC/MS $100 per sample
Tissue Imaging and Biofluids Core Personnel
L to R: Julie Coleman, Michele Reyzer,PhD;, Olivia Faison, Erin Seeley,PhD, and Lisa Zimmerman, PhD.
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 Imaging is 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-MSgenerates 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.
High-dose infection(abscesses present)
Uninfected(abscesses absent)
Low-dose infection(abscesses absent)
m/z 10,165 m/z 12,135
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.
Example of data generated using Tissue/Biofluids Core Facility
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