Mass Spectrometry atThe University of Louisville HSC

Jon Klein, M.D., Ph.DDirector

Michael L. Merchant, Ph.D.Technical Director, Proteomics Laboratory

Department of Medicine

Background

• Biomolecular Mass Spectrometry Core – Established by Bill Pierce ~1997

• Proteomics Core – Established 1998• Merger of both laboratories into a single HSC

Mass Spectrometry Core – 2012

Working definitions• Proteome

– the total set of proteins expressed in a biological compartment at a given point in time. Now recognized to include details of quantity and extent of modifications.

• Proteomics– The study of the proteome.

• The evolution of proteomic research follows three epochs of mass spectrometry development-– First generation (MS) and peptide mass fingerprinting (PMF)– Second generation and use of tandem mass spectrometry (MS/MS

or MS2) to obtain amino acid sequence– Third generation and the development of MSn methods for

quantitative and advanced qualitative proteomic data sets

Expression and quantitative proteomics•Define and quantify the protein components:

Functional proteomics.•Define the interactions among proteins:

Protein Interaction Networks (PIN)•Define the mechanisms by which proteins communicate with each other:

Protein Signaling Networks (PSN)

Three Broad Categories of Proteomic Studies

Courtesy of K. McLeish (2012)

• Define the question proteomics will answer• Isolation of protein-containing structure• Protein extraction: sonication, chaotropes, detergents

• Protein separation: 2DE, HPLC, LC

• Protein digestion: trypsin vs Lys-C

• Mass spectrometry analysis• Quantification: label (SILAC, DIGE) or label-free (spectral

counting)

• Bioinformatics

Steps in Proteomic Analysis

Courtesy of K. McLeish (2012)

Basic Mass Spectrometer

WorkStation

Flight Path

Separation technologies

• Three common separation modalities– Electrophoresis– Chromatography– Affinity enrichment (e.g. immunoaffinity)

• Why do we need separations– To increase sensitivity for detection of lower

abundant proteins.

Most samples have dynamic range of protein expression that exceeds the detector on the mass spectrometer. Consider

plasma:

Anderson NL, Anderson NG, MCP 1(11) 845-869 2002

Combinations of chromatography and electrophoresis to help observe, identify

and quantify low abundant proteins

InertBead

chickenantibody

(IgY)

Antibodies developed to-

Whole sample in

Fractionated sample out

Low abundant proteins firstHigh abundant proteins last

Immunoaffinity removal of abundant proteins to reveal low abundant proteins

Add Antibody againstProtein of interest

Antibody binds toProtein of interest

Adding protein A makes antibody-protein complex insoluble Centrifugation of solution

Pellets the complex.Removal of supernatant and washing

Elute Protein Trypsin digestion

Loading on mass spectrometer

Acquire tandem-MS data

Search MS/MS data against

human protein database w/ or w/o –P modification

Peptideidentification

Immunoprecipitation and mass spectrometry analysis of associated-binding proteins

Courtesy of Madhavi Rane (2012)

Mass spectrometers

Best advice on what instrument to use is:Know your goal and collaborate with the MS lab that will select the correct approach for your goal.

Prevailing MS-based Proteomic Method

Effect of improved mass accuracy

High Resolution MS at the HSC

• Orbitrap LTQ Velos Elite Mass Spectrometer with ETD capability• Mass Accuracy <3ppm RMS with external calibration, <1ppm RMS using internal calibration• Purchased with VA ShEEP grant and additional funds from the EVPRI, SOM Dean, Department of Medicine and Division of Nephrology

CostsInstitution UofL MD

AndersonTexas A&M

UVA Dana-Farber(Harvard)

University of Albany

MALDI $30 $50 N/A $105 $50 $50

Peptide Mass Fingerprinting

$30 N/A $107* $380 $150 for first sample

N/A

Analysis of Complex mixture- 1D-ShotgunLTQ-Orbitrap-Elite

$566 $1,250* N/A N/A N/A $2,000

*Intramural pricing, price for extramural collaborators not listed

HSC Mass Spectrometry Core Team

• Michael Merchant• Jian Cai• Danny Wilkey• Ming Li

HSC Mass Spectrometry Core

• Goals– Provide innovative state of the art MS – Collaborate– Enhance competitiveness of single and multi-

investigator grant applications– Be as self-supporting as possible

Acknowledgements

• Bill Pierce• Russ Prough• Department of Veterans Affairs• CEGeMM/ Ron Gregg• Don Miller• Toni Ganzel

Kindly Do Not Forget That Today is “International Talk Like a Pirate Day”