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 Proteomics Core – Established 1998 Merger of both laboratories into a single HSC Mass Spectrometry Core – 2012

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

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 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 Ionization Source Mass Analyzer Detector Work Station 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

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

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)

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

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

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

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