Vanderbilt University Mass Spectrometry Core Services

Presentation Transcript

David L. Hachey, PhD

Director 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

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

J Compton

MS Engineer

G

Bowersock

Network Engineer

RM Caprioli

M Casey

Admin Officer

Director, MSRC

Research

Bioanalytical

Proteomics

Tissue Profiling

Maintenance

P Chaurand

DC Liebler

L Zimmerman

DL Hachey

Res Assist. Prof

Director

Assoc Co-Director

Assoc Co

Director

DS Cornett

DB Friedman

Res Instructor

M Reyzer

MW Calcutt

Assoc Co

-

Director

Asst. Director

Assoc Co-Director

J Mobley

A Ham

Res Instructor

Research

Assistants

Research

Assistants

G Wernecke

Assoc Co

-

Director

Software Systems

Research

Assistants

Research

Assistants

Postdoctoral

Fellows

Visiting

Scholars

Graduate

Students

…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.

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

0.1

0.05

Standardized Log Abundance

0

-0.05

-0.1

-0.15

0 h

8 h

24 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

…Proteomics Core LC/MS/MS

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 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.

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.