Transcript 2a Proteomics

AH Biology: Unit 1
Proteomics and Protein
Structure 1
Proteomics
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, eg lyzozymes found in lysosomes
and proteins required for organelles such as
mitochondria.
2. Membrane bound, eg intrinsic and extrinsic
proteins.
3. Extracellular, eg 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.
• 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.
2.
3.
4.
Sanger sequencing in detail
gel electrophoresis
cycle sequencing
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?