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Introduction to Proteomics

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What is Proteomics?

Proteomics - A newly emerging field of life science research that uses High Throughput (HT) technologies to display, identify and/or characterize all the proteins in a given cell, tissue or organism (I.e. the proteome).

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3 Kinds of Proteomics

Expressional Proteomics Electrophoresis, Protein Chips, DNA Chips, SAGE Mass Spectrometry, Microsequencing

Functional Proteomics HT Functional Assays, Ligand Chips Yeast 2-hybrid, Deletion Analysis, Motif Analysis

Structural Proteomics High throughput X-ray Crystallography/Modelling High throughput NMR Spectroscopy/Modelling

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Expressional Proteomics

2-D Gel QTOF Mass Spectrometry

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Why Expressional Proteomics?

Concerned with the display, measurement and analysis of global changes in protein expression

Monitors global changes arising from application of drugs, pathogens or toxins

Monitors changes arising from developmental, environmental or disease perturbations

Applications in medical diagnostics and therapeutic drug monitoring

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Expressional Proteomics

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Expressional Proteomics

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Examples Jungblut PR et al., “Proteomics in Human

Disease: Cancer, Heart and Infectious Disease” Electrophoresis 20:100-110 (1999)

Zhukov TA et al., “Discovery of distinct protein profiles specific for lung tumors and pre-malignant lung lesions by SELDI” Lung Cancer 40:267-279 (2003)

Ghaemmaghami S, et al., “Global analysis of protein expression in yeast” Nature 425:737-741 (2003).

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Functional Proteomics

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Why Functional Proteomics? Concerned with the identification and

classification of protein functions, activities locations and interactions at a global level

To compare organisms at a global level so as to extract phylogenetic information

To understand the network of interactions that take place in a cell at a molecular level

To predict the phenotypic response of a cell or organism to perturbations or mutations

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Functional Proteomics (in vitro)

• Multi-well plate readers• Full automation/robotics• Fluorescent and/or chemi-

luminescent detection• Small volumes (mL)• Up to 1536 wells/plate• Up to 200,000 tests/day• Mbytes of data/day

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Functional Proteomics

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Functional Proteomics

In silico methods (bioinformatics) Genome-wide Protein Tagging Genome-wide Gene Deletion or

Knockouts Random Tagged Mutagenisis or

Transposon Insertion Yeast two-hybrid Methods Protein (Ligand) Chips

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Examples

• Uetz P et al., “A Comprehensive Analysis of Protein-Protein Interactions in Saccharomyces cerevisiae” Nature 403:623-627 (2000)

• First example of whole proteome analysis• 957 putative interactions• 1004 of 6100 predicted proteins involved

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Examples• Huh, K et al., “Global analysis of protein

localization in budding yeast” Nature, 425:686-691(2003)

• Used a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein (GFP) fusion proteins

• Localized 75% of the yeast proteome, into 22 distinct subcellular localization categories

• Provided localization information for 70% of previously unlocalized proteins

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Examples

• Edwards JS & Palsson BO “Systems properties of the H. influenzae Rd metabolic genotype” J. Biol. Chem. 274:17410-17416 (1999)

• First example of metabolic/phenotypic prediction using proteome-wide information

• Converting sequence data to differential equations so as to predict biology/behavior

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Structural Proteomics

• High Throughput protein structure determination via X-ray crystallography, NMR spectroscopy or comparative molecular modeling

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Structural Proteomics:The Goal

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Why Structural Proteomics?

• Structure Function

• Structure Mechanism

• Structure-based Drug Design

• Solving the Protein Folding Problem

• Keeps Structural Biologists Employed :D

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Structural Proteomics: The Motivation

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The Protein Fold Universe

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Protein Structure Initiative

Organize all known protein sequences into sequence families

Select family representatives as targets

Solve the 3D structures of these targets by X-ray or NMR

Build models for the remaining proteins via comparative (homology) modeling

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Protein Structure Initiative

Organize and recruit interested structural biologists and structure biology centres from around the world

Coordinate target selection Develop new kinds of high

throughput techniques Solve, solve, solve, solve….

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Structural Proteomics - Status 20 registered centres (~30

organisms) 82700 targets have been selected 52705 targets have been cloned 29855 targets have been expressed 12311 targets are soluble 1493 X-ray structures determined 502 NMR structures determined 1743 Structures deposited in PDB

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Structural Proteomics - Status

543 structures deposited by Riken 265 structures deposited by Mid-West 187 structures deposited by North-East 179 structures deposited by New York 178 structures deposited by JCSG (UCSD) 52 structures deposited by Berkeley 31 structures deposited by

Montreal/Kingston

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Bioinformatics & Proteomics

Proteomics Genomics

Medicine

Bioinformatics

Agriculture

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Bioinformatics & Functional Proteomics

How to classify proteins into functional classes?

How to compare one proteome with another? How to include functional/activity/pathway

information in databases? How to extract functional motifs from

sequence data? How to predict phenotype from proteotype?

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Bioinformatics & Expressional Proteomics

How to correlate changes in protein expression with disease?

How to distinguish important from unimportant changes in expression?

How to compare, archive, retrieve gel data? How to rapidly, accurately identify proteins

from MS and 2D gel data? How to include expression info in databases?

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Bioinformatics & Structural Proteomics

How to predict 3D structure from 1D sequence? How to determine function from structure? How to classify proteins on basis of structure? How to recognize 3D motifs and patterns? How to use bioinformatics databases to help in

3D structure determination? How to predict which proteins will express well

or produce stable, folded molecules?