Proteomics (1)

Proteomics (1)

INTRO
1. The proteome can be described as the entire complement of proteins that is(or can be) expressed by a cell, tissue or organism
  1. the term was first used by Marc Wilkins, a PhD student, in 1994
2. challenges associated with studying proteomics
  1. proteins have diverse physio-chemical properties
    1. they've undergone different, separate post-translational modifications and alternative splicing
  2. Proteins have a dynamic range of expression
    1. also very structurally, temporally, and spatially dynamic
  3. Methods for protein analysis are complex and are still (rapidly) evolving
    1. relatively large amounts of sample required for analysis
      1. can be hard to obtain
        especially in large numbers necessary for clinical trials
    2. Data can take days to produce
      1. data analysis can be extensive and time consuming
3. benefits of proteomics
  1. focuses on gene products
    1. the active agents in cells/tissues/organisms
    2. mRNA expression analysis (DNA arrays and micrarrays) do not always reflect the expression level of proteins
      1. merely reflects proteins that are coded for
  2. Biological samples (CSF, urine, serum etc) used for proteomic analysis are not suitable for mRNA expression analysis
  3. Modifications of proteins that are not apparent from DNA sequencing, i.e. post-translational modifications can be analysed using proteomics
  4. Proteomics can be used to analyse the location of proteins
  5. Proteomics will ultimately lead to the determination of protein function and therefore, a more detailed understanding of biological systems
Methods
1. gel based
  1. Protein separation by 2-dimensional gel electrophoresis of intact proteins (DIGE)
    1. 2DE
      1. developed in the mid to late 1970's
      2. separates proteins based on their molecular weight and isoelectric point (pI)
      3. can detect proteins using a number of different dyes, including fluorescent ones
      4. there is a problem with protein identification however and so it must be used in conjunction with mass spec (MS)
        Pick a spot on the gel that you want to ID and then use an automated spot picker to select this for MS analysis
    2. 2DE DIGE
      1. staining technique using the gel being run and a 2nd control gel whereby a different dye is placed on each and the differences in spots are analysed when the two gels are superimposed onto each other
        used to determine if spot differences from conventional 2d are due to induced biological changes or differences in the way the gels have been run/cast/stained
2. MS-based "gel-free" methods
  1. "shotgun" LC-ESI-MS/MS of total enzymic digest of proteins - peptides
    1. Quantification by stable isotope labelling (e.g. ICAT, iTRAQ, SILAC) or label free
  2. Protein MS
    1. measures m/s (mass/charge ratio)
    2. protein/peptide in gas
    3. measure intact protein or peptide or 'fragmentation' in mass spectrometer
    4. Protein can then be identified based on the ms image produced of teh different amino acids contained within it
  3. MALDI-MS
    1. Advantages
      1. analyte deposited in solid form
      2. tolerant to salts
      3. produces mostly singly charged ions
    2. dissadvantages
      1. coupling to peptide fractionation
      2. produces mostly singly charged ions
  4. Electrospray Ionisation
    1. Analyte in aqueous solution sprayed through the bore of a needlie
    2. Formation of charged droplets occurs
    3. Desolvation of the droplets takes place in a heated capillary
    4. Ions are transferred from liquid to gaseous phase by increasing charge
3. Protein Chips/arrays
  1. intact proteins
  2. e.g. protein, tissue and antibody arrays
why perform proteomic analysis (rely on high quality of samples)
1. to provide a mechanistic insight into both the disease pathogenesis and the identification of drug targets
2. Markers:
  1. proteomic analysis is performed to provide markers for patient stratification, for unbiased discovery, diagnosis and prediction of response to treatment and monitoring of patient response to treatment.
  2. Also performed to find markers for drug development and use
proteomic biomarker discovery process
1. Discovery
  1. sample accrual
  2. proteomics discovery
    1. protein separation
    2. protein detection
    3. Protein characterisation and identification (MS)
  3. uses other analytes (anything measurable)
2. Confirmation Assay Development
  1. Antibody based
    1. Western blotting
    2. ELISA
  2. Mass Spectrometry based
    1. Multiple reaction monitoring (MRM)
  3. Multi-analyte assays
3. Validation/Qualification
  1. additional clinical samples
  2. Large multicentre cohorts
  3. Large scale clinical trials
  4. "Robust" high-throughput assays
4. Approval & Adoption
  1. Regulatory Authorities
  2. Clinician Adoption
  3. Impact measurement
  4. Clinical Assays

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