Center for Human Health and the Environment Proteomics Section Center for Human Health and the Environment Leader: Michael S. Bereman Thankyou for the introduction Will

Central Dogma of Biology DNA RNA Proteins Metabolites What can happen? What appears to be happening What makes it happen What has happened or is in process Environmental Factors Human Health and Disease

Systems Technology Core Molecular Services/Expertise with Integrated Bioinformatic Support

Why Measure Proteins? – Molecules That Do Work Christine Vogel and Edward M. Marcotte, Nature Review Genetics, 2012 Mouse fibroblast cells Intimately tied to cell state mRNA measurements are often not a true proxy Other information Protein complexes PTMs Protein structure Enzymes – control metabolic processes Structural molecules – provide structure Storage proteins Defensivie proteins – that render bacterial and viruses inactive Central dogma slide, showing transcription to mRNA, translation, amino acids, can miss stuff if you just measure mRNA or DNA, Want to identify and quantify proteins http://chemistry.gsu.edu/Glactone/PDB/Amino_Acids/aa.html

Why Measure Proteins? – Molecules That Do Work “Protein abundances correlate well between C. elegans and Drosophila Melanogaster - better than RNA abundance and protein abundance within each species“ Shrimpf et. al., PLoS Biol 7: e48. 2009

Proteomic Experiments “Proteomics is the identities, quantities, structures, and biochemical and cellular functions of all proteins in an organism, organ, or organelle, and how they vary in space, time, and physiological state” Mol. Cell Proteomics 1:763:780 2002 Discovery Proteomics Targeted Proteomics What Proteins are in my sample? Are Proteins A, B, C in my sample? Shotgun proteomics Relative or absolute quantitation GeLC MS/MS So if you were to bring me a plasma sample between a farmer before exposure and then right after working in the fields and ask me what proteins are in these samples and how do that differ? But if you were to bring me that sample plasma sample or a cell sample from a model cell line where you were interested a specific signal transduction pathway and there were 50 proteins you were interested in measuring over and over again in 100 different cell lines “Exploratory” investigations “Hypothesis” driven investigations

The Current State of Proteomics via LC MS/MS Ahrens et al., Nature Reviews, 2012 Created by searching the keyword “proteomics” in Web of Science

Mission of the Proteomics Section The goal of the Proteomics Section is to provide CHHE members with expertise in experimental design, sample collection, preparation, and analysis for successful implementation of proteomics research. The Proteomics Section will enhance the ability of scientists working in the field of EHS to identify and capitalize on emerging opportunities in systems biology and the growing area of toxicoproteomics. Toxicoproteomics, a subfield of both proteomics and toxicogenomics, uses both global and targeted protein methodologies to identify and characterize critical proteins/complexes/pathways/receptors that are affected by, or respond to, chemical and environmental exposures. The Proteomics Section will provide increased impact of CHHE members’ research results by: 1) correlating genome/epigenome studies with protein expression patterns; 2) discovering novel biomarkers of exposure in biological fluids and; 3) evaluating the impacts of exposure on key biological processes by characterizing protein expression, interaction, and modification using both in vitro cellular and in vivo animal models of exposure.

AIMS of the Proteomics Section AIM 1. Provide expertise in study design for proteomic experiments. Number of replicates Buffers Extraction Detergents AIM 2. Advance EHS research by implementing both discovery and targeted proteomic experiments Applications of discovery and targeted proteomics to problems in environmental health science AIM 3. Provide consultation for members with projects that would benefit from other analytical measurement technologies

Technologies/Capabilities State of the art LC MS/MS Technologies Quadrupole Orbitrap Triple Quadrupole Discovery Experiments Targeted Experiments Homemade source Ultra high pressure LC (14000 PSI) Ultra high pressure LC (14000 PSI) 240,000 resolving power <5 ppm mass measurement accuracy Low detection limits (detection of fmols of peptides) High sensitivity 5 orders of linear dynamic range Fast!

Technologies/Capabilities Linear quadrupole ion trap Technology Development Sample preparation New ionization techniques Quick analyses Targeted peptide work

Software, Computing, and Data Storage Proteome Discoverer 1.4 Skyline DesignEase Computing Dell 5790 24 core Workstation Storage 12 TB Network Attached Storage Device RAID 10 Chorusproject.org

Training Basics of contemporary proteomics Sample preparation Data interpretation What does your data mean and what it does not mean

Sample Capacity A single sample could take 4 hours of instrument time Not equipped to handle 10s to 100s of samples Purpose – To provide data for manuscripts and preliminary results for competitive proposal submissions for EHS grants

What Can Contemporary Proteomics Do? In cells and tissue – routinely identify 2 – 3k proteins in a single injection Sensitive and precise relative quantification of a select group of proteins (targeted proteomics) ID of post-translational modifications upon enrichment (phosphorylation ubiquitination, etc) 4. Identification of protein interactions (Co-IP) Advantages and limitations to all analytical methods!

Conclusions Proteomics via LC MS/MS is a powerful technology used to measure proteins in complex mixtures Proteomics Section Mission is to provide expertise in experimental design, sample preparation, and analyses to members of CHHE