THERE'S LITTLE DOUBT THAT PROTEOMICS -- THE STUDY OF AN ORGANISM'S COMPLETE COMPLEMENT OF PROTEINS -- WILL HAVE GREAT IMPACT IN ALL AREAS OF THE LIFE SCIENCES IN THE YEARS

TO COME. AND THE REASON IS CLEAR. "TO REALLY UNDERSTAND BIOLOGICAL PROCESSES, WE NEED TO UNDERSTAND HOW PROTEINS FUNCTION IN AND AROUND CELLS

SINCE THEY ARE THE FUNCTIONING UNITS," HANNO STEEN, DIRECTOR OF THE PROTEOMICS CENTER AT BOSTON CHILDREN'S HOSPITAL.

AH Biology: Unit 1 Proteomics and Protein Structure 1

Think

What is the proteome?What codes for the proteome?How will we figure out how the proteome works?Why is it important that we understand the proteome?What are the applications of this technology to

mankind in the future?

Proteomics

The proteome is the entire set of proteins expressed by a genome.

Activation and inactivation of genes

Transcription animation

Translation animation

RNA splicing

RNA splicing

When mRNA is transcribed in eukaryotic cells it is composed of introns and exons.

Introns are the non-coding sequence of the mRNA and will not be expressed in the protein molecule. They are spliced out (removed) from the mRNA.

Exons are the coding sequence and will be expressed in the protein molecule.

RNA splicing in detail.

Post-translational modification

Post-translational modification is the alteration of the protein after translation

Post-translational modification occurs in the rough endoplasmic reticulum, Golgi apparatus and target site of the protein.

Post-translational modification can involve

1. the addition of chemical groups

2. the covalent cleavage of the polypeptide

Post-translational modification

1. the addition of chemical groups that are catalysed by dedicated post-translational modification enzymes:

• phosphorylation (addition of a phosphate group) • acylation (addition of an acyl group RCO–, where R is an alkyl

group) • alkylation (addition of an alkyl group, eg methylation)• glycosylation (addition of a sugar group, eg glucose or

oligosaccharides)• oxidation.

2. the covalent cleavage of sections of the polypeptide

• proteases (trypsinogen to trypsin) • autocatalytic cleavage (the zymogen pepsinogen to pepsin).

Post-translational modification

These modifications give the proteins specific functions and target the proteins to specific areas within the cell and the whole organism.

1. Intracellular, e.g. lysosymes found in lysosomes and proteins required for organelles such as mitochondria.

2. Membrane bound, e.g. intrinsic and extrinsic proteins.

3. Extracellular, e.g. insulin and digestive enzymes.

Membrane proteins

Extracellular proteins and exocytosis

RNA splicing and post-translational modification

RNA splicing and post-translational modification results in the proteome being larger than the genome.

One gene may code for many proteins.

The proteome may be as many as three orders of magnitude larger than the genome.

Human genome = 30,000 genes approximately.

Human proteome > 100,000 proteins.

Regulation of gene expression

Because of regulation of gene expression not all genes are expressed as proteins in a particular cell.

The Jacob Monod hypothesis or lac Operon is an example of this process.

This ensures that the cell is energy efficient and producing proteins only when they are required.

the lac Operon and its control

Analysis of the genome

While DNA sequencing and microarray technology allow the routine analysis of the genome and transcriptome, the analysis of the proteome is far more complex.

Genome analysis involves the following techniques:

1. Sanger sequencing in detail2. gel electrophoresis3. cycle sequencing4. microarray in detail.

Analysis of the proteome

Proteome analysis involves:

1. Isolation of proteins expressed by an active cell at a given time.

2. The functional interaction between the proteins active in the cell.

Analysis of the proteome

Techniques used to identify expressed proteins:

1. 2D electrophoresis to separate out proteins from cell samples according to their charge (isoelectric point: pH at which the protein has no net charge and does not migrate in an electric field) and molecular weight (SDS PAGE).

2. Western blotting: Transfer proteins to nitrocellulose paper. Expose proteins to specific antibody coupled to a radioisotope, easily detectable enzyme or fluorescent dye. Identify desired protein/proteins.

3. Mass spectrometry to separate out proteins and identify specific fragments.

Analysis of the proteome

This is a complex process as the proteins expressed differ from cell to cell and within the life cycle of the cell.

In a multicellular organism all the different cell types throughout the lifetime of the organism would have to be sampled in order to determine all the possible proteins expressed.

Proteomics technologies and cancer.

SDS PAGE

SDS PAGE

Isoelectric point

Isoelectric point: pH at which the protein has no net charge and does not migrate in an electric field.

Western blotting

Protein structure and activity

The distinguishing feature of protein molecules is their folded nature and their ability to bind tightly and specifically to other molecules.

Enzymes and the induced fit to their substrate is an example of this.

The binding of oxygen to haemoglobin also illustrates this principle.

Mass spectrometry

For more detail on mass spectrometry click the following link to Leeds University.

Enzymes induced fit

Haemoglobin

Binding and conformational change

Binding causes a conformational change in the protein, which may result in an altered function and may be reversible. Enzyme inhibition Sodium potassium pump Cell proliferation and phosphorylation

Proteins may have one or more stable conformations depending on binding.

This allows the property, regulation and activity of the protein to be controlled.

The proteasome animation.

Proteomics: further reading

Boston Children’s Hospital: Interactive guide to sequencing and identifying proteins.

Read the following journals to see how proteomics is used. These journals will form the basis for Proteomics Tutorials 1 and 2.

Knight JDR, Qian B, Baker D, Kothary R (2007) Conservation, Variability and the Modeling of Active Protein Kinases. PLoS ONE 2(10): e982. doi:10.1371/journal.pone.0000982.

Roy N, Nageshan RK, Pallavi R, Chakravarthy H, Chandran S, et al. (2010) Proteomics of Trypanosoma evansi Infection in Rodents. PLoS ONE 5(3): e9796. doi:10.1371/journal.pone.0009796.

Think

What is the proteome?What codes for the proteome?How will we figure out how the proteome works?Why is it important that we understand the

proteome?What are the applications of this technology to

mankind in the future?