mass spectrometry at the university of louisville hsc jon klein, m.d., ph.d director michael l....
TRANSCRIPT
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)
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.
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
• 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