Proteomics

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Proteomics

• Alice Skoumalová

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• What is proteomics?
• The large-scale study of proteins

Proteomics
Genomics
PROTEingenOME
Expression
Genom
Proteom
posttranslational modification alternative
splicing alternative folding
All proteins produced by an organism The human
body contains millions proteins One organism has
different protein expression in different parts
of its body, stages of its life cycle and
environmental conditions
All genes in DNA of an organism The human genome
contains 20-25000 genes The genom is a constant
entity
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• Increase in protein diversity
• Posttranslational modification
• Alternative splicing
• Alternative folding
• Primary transcript
• mRNA before the posttranscriptional modification

Alternative splicing
Posttranslational modification
Alternative folding
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• Posttranslational modification
• The chemical modification of a protein after its
  translation
• Addition of functional groups (acetate,
  phosphate, lipids, carbohydrates)
• Modification of amino acids
• Structural changes ( the formation of disulfide
  bridges, proteolytic cleavage)

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Alternative splicing of a pre-mRNA transcribed
from one gene can lead to different mature mRNA
molecules and therefore to different proteins
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Alternative folding The protein folding proceeds
from a disordered state to progressively more
ordered conformations corresponding to lower
energy levels
Local minimum (alternative conformation)
Global minimum (native state)
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Basic proteomic analysis scheme
Protein mixture
1. Separation 2D-PAGE
Individual proteins
2. Spot cutting Trypsin digestion
Peptides
3. Mass analysis Mass spectroscopy
4. Sequence analysis Peptide fragmentation
Peptide mass
Sequence information
5. Database search
Protein identification
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• 2D gel electrophoresis
• Proteins are separated correspond to their charge
  (isoelectric point) in first dimension and their
  mass (migration) in second dimension
• The separation by isoelectric point (pI) in the
  pH gradient
• At all pHs other than their isoelectric point,
  proteins will be charged. If they are positively
  charged, they will be pulled towards the more
  negative end of the gel and if they are
  negatively charged they will be pulled to the
  more positive end of the gel. The proteins will
  accumulate at their isolelectric point
• 2. The separation by mass in polyacrylamide gel
  (PAGE)
• An electric potential is applied, at a 90
  degree angle from the first field. The proteins
  move proportionally to their mass (the gel acts
  like a molecular sieve).
• The result of this is a gel with proteins
  spread out on its surface.
• 3. Staining
• Silver or coomassie

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2D gel electrophoresis The synchronous analysis
of hundreds or even thousands of
proteins Proteins spread out on the surface
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• Mass spectroscopy (MALDI-TOF)
• Petides are analysed by MALDI-TOF mass
  spectrometer (Matrix-assisted laser
  desorption/ionization time-of-flight)
• A peptide is placed on a matrix, which causes the
  peptide to form crystals
• Then the peptide is ionized with a laser beam and
  an increase in voltage at the matrix is used to
  shoot the ions toward a detector in which the
  time it takes an ion to reach the detector
  depends on its mass
• The higher the mass, the longer the time of
  flight of the ion
• Peptide mass fingerprinting (PMF)
• Founded masses are compared to the genome
• Computer programs translate the known genome into
  proteins, then theoretically cut the proteins
  into peptides, and calculate the masses of the
  peptides
• They then compare the masses of the peptides of
  the unknown protein to the theoretical peptide
  masses of each protein encoded in the genome.

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• Application of proteomics in medicine (disease
  proteomics)

The role of proteins in the pathogenesis of
diseases
Protein expression in diseases
Using specific protein biomarkers to diagnose
disease Alzheimer disease (amyloid ß) Heart
disease (interleukin-6 and 8, serum amyloid A,
fibrinogen, troponins) Renal cell carcinoma
(carbonic anhydrase IX)
Biomarkers of diseases
Design of new drugs
Information about proteins causing diseases is
used for the identification of potential new drugs
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The role of proteins in the pathogenesis of
diseases

• Alzheimer disease (AD)
• Age-related neurodegenerative disorder
  characterized by neuronal death and loss of
  synaptic connections
• Neuropathologically is defined by the
  accumulation of two types of insoluble fibrous
  material, amyloid-ß in senile plaques and
  intracellular neurofibrillary tangles

Oxidative stress Protein oxidation
Oxidatively modified proteins
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Protocol for the identification of oxidized
proteins by proteomics
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The role of proteins in the pathogenesis of
diseases

• Results
• Confirmation of the role of oxidative stress in
  Alzheimer disease
• Posttranslational modification of brain proteins
  caused by oxidative damage is involved in the
  pathogenesis of AD

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Biomarkers of diseases

• Proteome-based plasma biomarkers for AD
• Diagnosis of AD
• On clinical groundspost mortem (histology)
• There is no reliable diagnostic test
• Plasma may offer a rich source of disease
  biomarkers

• Identification of diagnostic biomarkers in the
  blood by proteomics
• Plasma samples of patients and control were
  analysed by 2D gel electrophoresis
• Spots that were significantly different between
  case and control groups were excised and analysed
  by mass spectroscopy

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• Results
• 15 spots were significantly different between
  patients and controls
• MS analysis ?2-macroglobulin, complement factor
  H,

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Design of new drugs
The proteome information identifies proteins
associated with a disease 1. Computer software
can use these proteins as targets for new drugs
For example, if a certain protein is implicated
in a disease, the 3D structure of that protein
provides the information a computer program needs
to design drugs to interfere with the action of
the protein (a molecule that fits the active site
of an enzyme, will inactivate the enzyme) 2. As
genetic differences among individuals are found,
researchers will use these same techniques to
develop personalized drugs that are more
effective for the individual
Virtual ligand screening
The identification of new drugs to target and
inactivate the HIV-1 protease (cleaves a very
large HIV protein into smaller, functional
proteins virus cannot survive without this
enzyme it is one of the most effective protein
targets for killing HIV)
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Questions

• An increase in protein diversity is due to
• Proteins can be separated by
• Proteins and peptides are indentified by
• Identification of the renal carcinoma biomarkers
  in the plasma
• Using of computer sofware for the development of
  new drugs

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• Summary
• Proteomics studies proteins, particularly their
  structure, function and interaction
• The genome has already been analysed, now
  scientists are interested in the human proteome
  (millions of proteins)
• Key technologies used in proteomics are 2D gel
  electrophoresis and mass spectrometry
• Proteins play a central role in the life of an
  organism, their malfunction startes diseases
  proteomics is instrumental in discovery of
  pathogenesis of disease, biomarkers and
  potential therapetic agents