Slide 1

Introduction to Proteomics
Paul D. Brown, PhD paul.brown@uwimona.edu.jm BC34B: Advanced Biochemistry Lecture slides on OurVLE

Slide 2

The -omics and -ome Concepts

-omics area of research Genome
derived from German Genom 1920; Hans Winkler Haploid chromosome set of an organism or species

-om or -ome
Coined by German and French cytologists to signify cellular organelles collectively Mitochondria (chondriome); vacuoles (vacuome)

English names of sciences: -ics

16th Century Hence, economics, mathematics, optics

Slide 3

Genomics and Proteomics: new fields with a new vocabulary

Genomics
DNA Genome

RNA

Transcriptome

Proteomics
Proteins Proteome

Metabolites

Metabolome

Systems Biology
Protein-protein, Protein-DNA, Protein-RNA interactions

Interactome

Slide 4

Protein Chemistry/Proteomics Protein Chemistry

Individual proteins Complete sequence analysis Emphasis on structure and function Structural biology

Proteomics
Complex mixtures Partial sequence analysis Emphasis in identification by database matching System biology

Slide 5

Slide 6

Why are we studying proteins?

Proteins are the mediators of functions in the cell Deviations from normal status denotes disease Proteins are drug/therapeutic targets

Slide 7

Proteomics and biology /Applications

Protein Expression Profiling Proteome Mining
Identifying as many as possible of the proteins in your sample Identification of proteins in a particular sample as a function of a particular state of the organism or cell

Posttranslational modifications
Identifying how and where the proteins are modified

Functional proteomics Protein-protein interactions Protein-network mapping Structural Proteomics

Determining how the proteins interact with each other in living systems

Protein quantitation or differential analysis

Slide 8

Defining Proteomics
Branch of discovery science focusing on proteins Proteome

In 1994, defined as the complete set of proteins that is expressed and modified following expression by the entire genome in the lifetime of a cell Can be more specific: the complement of proteins expressed by a cell at any one time

Today proteomics is a scientific discipline that will bridge the gap between our understanding of genome sequences and cellular behaviour.

Slide 9

The Proteome
Dependent on environmental factors, disease, drugs, stress, growth conditions Understanding the proteome:
Cycle of proteins Proteins as modular structures Functional families Genomic sequences Protein expression/Protein levels

Slide 10

Life cycle of a protein

Information found in DNA is used for synthesis of the proteins
mRNA

Protein

Folding

Translocation
to specific subcellular or extracellular compartments

Posttranslational Processing
Proteolytic Cleavage Degradation Damage
-free radicals

Acylation Methylation Phosphorylation Sulfation Selenoproteins

Environmental
-chemicals radioactiivty

Ubiquination Glycolysation

Slide 11

Molecular Structures
Primary structure a chain of amino acids Secondary structure three dimensional form, formally defined by the hydrogen bonds of the polymer
Amino acids vary in their ability to form the various secondary structure elements. Amino acids that prefer to adopt helical conformations in proteins include methionine, alanine, leucine, glutamate and lysine (MALEK) The large aromatic residues (tryptophan, tyrosine and phenylalanine) and Cbranched amino acids (isoleucine, valine and threonine) prefer to adopt -strand (sheet/pleat) conformations.

Slide 12

Molecular Structures Tertiary structure the overall 3-D shape of the protein (fold)

The 3-D shape might be critical for function. For example, specific binding sites for substrates on enzymes Specific sequences that also confer unique properties and functions, motifs or domains

Quaternary structure formation usually involves the "assembly" or "coassembly" of subunits that have already folded

Slide 13

Sequence alignment
A way of arranging primary sequences (of DNA, RNA, or proteins) in such a way as to align areas sharing common properties.

The degree of relatedness, similarity between the sequences is predicted computationally or statistically ClustalW BLAST

Software tools used for general sequence alignment tasks include:

Slide 14

Functional families Proteins can be grouped into functional families (proteins that carry out related functions)
Structural Signaling pathways Metabolic Transportation

3% Ribosomal 4%

Hypothetical Channels 1%

Factors 4%

Enzymes 45%

Structural 9% Other 30% Heat Shock 4%

Slide 15

Sequence-Structure-Function

Homology searching (BLAST)

Sequence
Structure Function

Threading

Slide 16

Genomic sequencing/ Protein level

Genome size (bp) 5,386 580,000 12.1 106 3.2 109 90 109 670 109 Organism X-174 virus Mycoplasma genitalium Yeast (S. cerevisiae) Human Lilium longiflorum Amoeba dubia

Slide 17

Proteome complexity

Slide 18

Protein Heterogeneity
See much larger number of spots compared to protein species they represent
H.influenzae: 1500 spots represent 500 different proteins

More than 100 modification forms known A single protein may carry several modifications Modified proteins show different properties compared to unmodified counterparts In most cases, we do not know the origin or the biological significance of the observed heterogeneities

Slide 19

2D gel image of brain proteins

-enolase

A Partial 2D-gel images showing enolase from human brain. The protein is represented by one spot when IEF was performed on pH 3-10 non-linear IPG strips (A), and by six spots when IEF was performed on pH 4-7 strips (B).
About 3000 Spots after Coomassie Stain Electrophoresis, 1999, 20 (14) 2970

Slide 20

Protein Heterogeneity

http://www.lcb.uu.se/course/embo2001/binz/presentation-PAB-intro/ppframe.htm

Slide 21

Protein level The level of any protein in a cell at a given time:

Transcription rate Efficiency of translation in the cell The rate of degradation of the protein

Expression profiling
What genes are expressed in a particular cell type of an organism, at a particular time, under particular conditions Is the amount of mRNA correlated to gene expression?

Slide 22

Types of proteomics
Expression proteomics
Quantitative maps of proteins expressed from cells or tissues Mainstay, 2D-GE (IEF followed by SDSPAGE) Applications:

Co-expression as a means of identifying biological pathways and their perturbation by disease (Target validation and development) Drug action and effects of biological stimuli Identification of disease markers

Slide 23

Types of proteomics
Cell-map (Interaction) proteomics
Physical maps of protein cellular location and interactions Applications:
Assignment of protein function Identification of protein networks Target validation

Methods:
Yeast-2-hybrid system Protein affinity chromatography (ligand binding)

Slide 24

Types of proteomics
Structural proteomics
Structural maps of protein and protein domain 3D conformation Applications

Structure-function relationships Prediction of 3D structure from genomic sequence Drug discovery Identification of structurally-related orthologues

Slide 25

Large scale proteomics studies do Data not increase our knowledge significantly Knowledge
2000-present

Single protein studies

Targeted proteomics studies Proteomics studies

1-10 Number of genes

10-100

30,000 (genomes)

Slide 26

Yeast Interactome
Ho et al. Nature (2002) 415: 180-183

Targeted Proteomics on 725 proteins representing 10% of yeast genome Detection of 3617 Proteinprotein interactions covering 25% of the genome

Slide 27

Knowledge from proteomics studies is limited by our inability to analyze efficiently large data sets
Gene name Interaction

Proteomics studies highlight the extreme complexity of interactions in a genomic scale.

Proteomics is facing the challenge of analyzing large and highly complex and very noisy data sets. Bioinformatics is integrated in proteomics projects to mine data and is becoming more and more important.

Slide 28

How does Proteomics fit in with other Functional Genomic approaches?

Genomic motifs

Genome Sequence (2% coding)

cDNA and ESTs databases

Gene Identification
Comparative genomic approaches

Protein identification

Gene Expression
cDNA Expression profiling SAGE Protein expression profiling

Gene Function
Co-expression Transgenesis Gene trapping Global chemical mutagenesis Naturally-occurring mutations

How well do mRNA levels reflect the abundance of their respective proteins?

Protein PTMs Protein Cellular location Protein Interactions and networks Protein 3D structure

Slide 29

Basic approaches for proteome analysis

Proteome
Directly Indirectly

Tissues cell lines, serum, urine, etc.

Expression Libraries

Specific Cell types Laser capture Microdissection

Subcellular fractions Density gradient centrifugation

Multiprotein complexes Co-immunoprecipitation

Identify Protein-protein and Ligand Interactions Combinatory peptides (phage display libraries)

Protein Resolution 2D gel electro., Liquid Chromat., Protein Arrays and Chips Quantitation Phosphorimaging Densitometry Fluorography Identification Characterization Mass spectrometry Microsequencing

Epitope tags and GST fusion proteins; Yeast 2 hybrid

Slide 30

How does proteomics help to identify genes involved in important diseases?

An example in Human Genetics

Slide 31

Genomics Databases can identify the candidate genes involved in human diseases
More than one candidate gene

(NCBI) National Center for Biotechnology Information

SNPs Single Nucleotide Polymorphism

Slide 32

Further analysis can identify the gene involved in a particular human disease
2D gel Only one candidate gene

Disease Gene Identified with mutations

Expression analysis

Microarray Gene expression data