1. 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

2. 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

3. Systems Technology Core
Molecular Services/Expertise with Integrated Bioinformatic Support

4. 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

5. 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: e

6. 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:
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

7. 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

8. 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.

9. 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

10. 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!

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

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

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

14. 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

15. 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!

16. 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