Lecture 19 – Functional Genomics - Proteomics Based on chapter 8 Functional and Comparative...

Lecture 19 –Functional Genomics - Proteomics

Based on chapter 8 Functional and

Comparative Genomics and web materials

Copyright © 2010 Pearson Education Inc.

Contents

1. Definition & Goals of proteomics

2. Proteomics technologies

a. 2-D gel electrophoresis

b. Mass spectrometry

c. Protein chips

d. Yeast two-hybrid method

e. Protein localization

3. Using proteomics to uncover transcriptional networks

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

What is proteomics?

1. A catalog of all proteins expressed throughout the life cycle of the organism.

2. A catalog of all proteins expressed under all conditions in an organism.

3. A catalog of all proteins expressed in all tissues of an organism.

The Goals of Proteomics are:

1. To catalog all proteins found in organisms.

2. To understand the function of all proteins in an organism

3. To understand how proteins of an organims interact with each other and where they localize in the cell.

The challenges of proteomics

1. Splice variants create an enormous diversity of proteins

a. ~25,000 genes in humans give rise to 200,000 to 2,000,000 different proteins

b. Splice variants may have very diverse functions

2. Proteins expressed in an organism will vary according to age, health, tissue, and environmental stimuli

3. Proteomics requires a broader range of technologies than genomics


Diversity of function in splice variants

1. Example: the calcitonin gene (Review of eukaryotic transcription regulation)

a. Gene variant #1i. Protein: calcitoninii. Function:

increases calcium uptake in bones

b. Gene variant #2i. Protein: calcitonin

gene-related polypeptide

ii. Function: causes blood vessels to dilate


Posttranslational modifications

1. Proteolytic cleavage

a. Fragmenting protein

b. Examplesa. Insulin

b. Trypsin

c. See previous slide

2. Addition of chemical groups


Chemical modifications

1. Phosphorylation: activation and inactivation of enzymes2. Acetylation: protein stability, used in histones3. Methylation: regulation of gene expression4. Acylation: membrane tethering, targeting5. Glycosylation: cell–cell recognition, signaling6. Hydroxyproline: protein stability, ligand interactions7. Ubiquitination: destruction signal8. Others

1. Sulfation: protein–protein and ligand interactions2. Disulfide-bond formation: protein stability3. Deamidation: protein–protein and ligand interactions4. Pyroglutamic acid: protein stability5. GPI anchor: membrane tethering6. Nitration of tyrosine: inflammation


Practical applications of Proteomics

1. Comparison of protein expression in diseased and normal tissues

a. Likely to reveal new drug targetsi. Today ~500 drug targetsii. Estimates of possible drug targets: 10,000–

20,0002. Protein expression signatures associated with drug

toxicitya. To make clinical trials more efficientb. To make drug treatments more effective


Technologies for proteomics

1. 2-D gel electrophoresisa. Separates proteins in a mixture on the basis of

their molecular weight and charge2. Mass spectrometry

a. Reveals identity of proteins3. Protein chips

a. A wide variety of identification methods4. Yeast two-hybrid method

a. Determines how proteins interact with each other

5. Biochemical genomicsa. Screens gene products for biochemical activity


2-D gel electrophoresis

1. Polyacrylamide gel2. Voltage across both

axesa. pH gradient along

first axis neutralizes charged proteins at different places

b. pH constant on a second axis where proteins are separated by weight

3. x–y position of proteins on stained gel uniquely identifies the proteins

BasicAcidicH

igh

MW

Lo

w M

W


Differential in gel electrophoresis

1. Label protein samples from control and experimental tissuesa. Cy3 for controlb. Cy5 for

experimental sample

2. Mix protein samples together

3. Identify identical proteins from different samples by dye color

withbenzoicacidCy3

withoutbenzoicacidCy5


Caveats associated with 2-D gels

1. Poor performance of 2-D gels for the following:

a. Very large proteins

b. Very small proteins

c. Less abundant proteins

d. Membrane-bound proteins

2. Presumably, the most promising drug targets


Mass spectrometry

1. Measures mass-to-charge ratio

2. Components of mass spectrometer

1. Ion source

2. Mass analyzer

3. Ion detector

4. Data acquisition unit

A mass spectrometer


Ion sources used for proteomics

Proteomics requires specialized ion sources

Electrospray Ionization (ESI)

Matrix-assisted laser desorption/ionization (MALDI)

ESI

MALDI


Mass analyzers used for proteomics

Detection methods Ion trap Time of flight (TOF)

Ion Trap

Time of Flight

Detector


A mass spectrum


Identifying proteins with mass spectrometry

1. Preparation of protein sample

a. Extraction from a gel

b. Digestion by proteases — e.g., trypsin

2. Mass spectrometer measures mass-charge ratio of peptide fragments

3. Identified peptides are compared with database

a. Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database

b. Match of experimental readout to database PMF allows researchers to identify the protein


Stable-isotope protein labeling

1. Stable isotopes used to label proteins under different conditions

2. Variety of labeling methodsa. Enzymaticb. Metabolicc. Via chemical

reaction3. Relative abundance of

labeled and nonlabeled proteins measured in mass spectrum


Limitations of mass spectrometry

1. Not very good at identifying minute quantities of protein

2. Trouble dealing with phosphorylated proteins

3. Doesn’t provide concentrations of proteins

4. Improved software eliminating human analysis is necessary for high-throughput projects


Protein chips

1. Thousands of proteins analyzed simultaneously

2. Wide variety of assays

a. Antibody–antigen

b. Enzyme–substrate

c. Protein–small molecule

d. Protein–nucleic acid

e. Protein–protein

f. Protein–lipid

Yeast proteins detectedusing antibodies


Fabricating protein chips

1. Protein substratesa. Polyacrylamide or

agarose gels

b. Glass

c. Nanowells

2. Proteins deposited on chip surface by robots


Difficulties in designing protein chips

1. Unique process is necessary for constructing each probe element

2. Challenging to produce and purify each protein on chip

3. Proteins can be hydrophobic or hydrophilic

a. Difficult to design a chip that can detect both


Lecture 19 – Functional Genomics - Proteomics Based on chapter 8 Functional and Comparative...

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