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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. Background. Biomolecular Mass Spectrometry Core – Established by Bill Pierce ~1997

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Mass spectrometry at the university of louisville hsc

Mass Spectrometry atThe University of Louisville HSC

Jon Klein, M.D., Ph.D

Director

Michael L. Merchant, Ph.D.

Technical Director, Proteomics Laboratory

Department of Medicine


Background
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
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 MSnmethods for quantitative and advanced qualitative proteomic data sets


Three broad categories of proteomic studies
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)


Steps in proteomic analysis
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)


Basic mass spectrometer

Work

Station

Basic Mass Spectrometer

Flight Path


Separation technologies
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


Whole sample in observe, identify and quantify low abundant proteins

chicken

antibody

(IgY)

Immunoaffinity removal of abundant proteins to reveal low abundant proteins

Inert

Bead

Fractionated sample out

Low abundant proteins first

High abundant proteins last

Antibodies developed to-


Immunoprecipitation and mass spectrometry analysis of associated-binding proteins

Adding protein A makes antibody-protein complex

insoluble

Add Antibody against

Protein of interest

Centrifugation of solution

Pellets the complex.

Removal of supernatant and washing

Antibody binds to

Protein of interest

Search MS/MS data against

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

Acquire tandem-MS data

Elute Protein Trypsin digestion

Peptide

identification

Loading on mass spectrometer

Courtesy of Madhavi Rane (2012)


Mass spectrometers

Mass spectrometers associated-binding proteins

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
Prevailing MS-based Proteomic Method associated-binding proteins


Effect of improved mass accuracy
Effect of improved mass accuracy associated-binding proteins


High resolution ms at the hsc
High Resolution MS at the HSC associated-binding proteins

  • OrbitrapLTQ 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


Costs
Costs associated-binding proteins

*Intramural pricing, price for extramural collaborators not listed


Hsc mass spectrometry core team
HSC Mass Spectrometry Core Team associated-binding proteins

  • Michael Merchant

  • Jian Cai

  • Danny Wilkey

  • Ming Li


Hsc mass spectrometry core
HSC Mass Spectrometry Core associated-binding proteins

  • 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
Acknowledgements associated-binding proteins

  • Bill Pierce

  • Russ Prough

  • Department of Veterans Affairs

  • CEGeMM/ Ron Gregg

  • Don Miller

  • Toni Ganzel



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