Vanderbilt-2006-07-28

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Vanderbilt UniversityMass Spectrometry Core

Services

David L. Hachey, PhD

Director of Mass SpectrometryProfessor of Pharmacology,

Biochemistry, Chemistry

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…An Overview

• Mass Spectrometry Research Center – A comprehensive, university-wideresearch facility dedicated to the development of advanced mass spectrometrictechniques for the study of complex biological systems.

• Research Core – Development of advanced mass spectrometric techniques forthe study of complex biological systems.

• The Metabolomics & Mass Spectrometry Core – An open-access servicefacility for qualitative and quantitative analysis of low molecular weight (<2,500Da) biomolecules.

• The Proteomics Core – A specialized service facility for the identification andcharacterization of proteins and protein complexes.

• Tissue Protein Profiling Core – A specialized service facility for profiling ofproteins in tissues and biological fluids.

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Mass Spectrometry

MS Core David HacheyWade Calcutt

Proteomics Dan Liebler

David Friedman

Amy Hamm

ResearchRichard CaprioliPierre Chaurand

Shannon Cornet

LieblerCaprioli

Hachey

BiomarkersLisa ZimmermanMichelle Reyzer

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RM Caprioli Director, MSRC

RM Caprioli Director, MSRC

DL Hachey

Director

M Casey Admin Officer

M Casey Admin Officer

MW Calcutt

Asst. Director

J Compton MS Engineer

P Chaurand Res Assist. Prof

J Mobley Res Instructor

PostdoctoralFellows

Graduate

Students

Research

Assistants

Visiting

Scholars

DC Liebler Director Director

L Zimmerman Assoc Co

L Zimmerman Assoc Co-Director

Research Bioanalytical Proteomics

Research

Assistants

DS Cornett Res Instructor

A Ham Assoc Co - Director

A Ham Assoc Co - Director

DB Friedman

Assoc Co - Director

DB Friedman

Assoc Co - Director

G Bowersock

Network Engineer

G Bowersock

Network Engineer

M Reyzer

Research

Assistants

Tissue Profiling Maintenance

Research

Assistants

G Wernecke

Assoc Co-Director

Software Systems

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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)

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…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

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…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 offacility instruments

• to establish a library of core analytical techniques for general use• to assist in developing new analytical methods

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…Core Operations - Techniques & Services

Structural Analysis of Biomolecules

- Identification of post translational modifications of proteins andDNA 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

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Lauren Beihoffer Betty Fox Kerry Fillgrove

July, 2004

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…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

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…Instrumentation & Facilities

GC/MS

Hewlett-Packard (Agilent) - 5989AThermoElectron (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.

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…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

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Proteomics Core

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MALDI-TOF MS and TOF/TOF

MS/MS on targeted proteins

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…Proteomics Core LC/MS/MS

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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 gel2D 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

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Tissue Imaging and Biofluids Core Personnel

L to R: Julie Coleman, Michele Reyzer,PhD;, Olivia Faison, Erin Seeley,PhD, and Lisa Zimmerman, PhD.

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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 isoften to discover patterns in the protein profiles of the samples that can classify thesamples 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 thegoal is to obtain a relatively high resolution image showing the distribution of variousproteins 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 todifferentiate between disease states.

Multidimensional Analysis (LC-MS-MS) of Biofluidscomprehensive analysis of fluids providing inventory of protein identified insamples.

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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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Documents

Vanderbilt-2006-07-28