1. Mass Spectrometry at The University of Louisville HSC
Jon Klein, M.D., Ph.D
Director
Michael L. Merchant, Ph.D.
Technical Director, Proteomics Laboratory
Department of Medicine

2. 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

3. Working definitions Proteome Proteomics
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

4. Three Broad Categories of Proteomic Studies
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)
Courtesy of K. McLeish (2012)

5. Steps in Proteomic Analysis
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
Courtesy of K. McLeish (2012)

6. Basic Mass Spectrometer
Ionization Source
Mass Analyzer
Detector
Work
Station
Flight Path

7. 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.

8. 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)

9. Combinations of chromatography and electrophoresis to help observe, identify and quantify low abundant proteins

10. Inert
Bead
chicken
antibody
(IgY)
Whole sample in
Immunoaffinity removal of abundant proteins to reveal low abundant proteins
Antibodies developed to-
Fractionated sample out
Low abundant proteins first
High abundant proteins last

11. Immunoprecipitation and mass spectrometry analysis of associated-binding proteins
Add Antibody against
Protein of interest
Antibody binds to
Adding protein A makes antibody-protein complex
insoluble
Centrifugation of solution
Pellets the complex.
Removal of supernatant and washing
Acquire tandem-MS data
Search MS/MS data against
human protein database w/ or w/o –P modification
Elute Protein Trypsin digestion
Peptide
identification
Loading on mass spectrometer
Courtesy of Madhavi Rane (2012)

12. 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.

13. Prevailing MS-based Proteomic Method

14. Effect of improved mass accuracy

15. 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

16. Costs Institution UofL MD Anderson Texas A&M UVA Dana-Farber (Harvard)
University of Albany
MALDI
$30
$50
N/A
$105
Peptide Mass Fingerprinting
$107*
$380
$150 for first sample
Analysis of Complex mixture- 1D-Shotgun
LTQ-Orbitrap-Elite
$566
$1,250*
$2,000
*Intramural pricing, price for extramural collaborators not listed

17. HSC Mass Spectrometry Core Team
Michael Merchant
Jian Cai
Danny Wilkey
Ming Li

18. 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

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

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