Proteomics (1)

Proteomics (1)

1. INTRO

   • The proteome can be described as the entire complement of proteins that is(or can be) expressed by a cell, tissue or organism
     • the term was first used by Marc Wilkins, a PhD student, in 1994
   • challenges associated with studying proteomics
     • proteins have diverse physio-chemical properties
       • they've undergone different, separate post-translational modifications and alternative splicing
     • Proteins have a dynamic range of expression
       • also very structurally, temporally, and spatially dynamic
     • Methods for protein analysis are complex and are still (rapidly) evolving
       • relatively large amounts of sample required for analysis
         • can be hard to obtain
           • especially in large numbers necessary for clinical trials
       • Data can take days to produce
         • data analysis can be extensive and time consuming
   • benefits of proteomics
     • focuses on gene products
       • the active agents in cells/tissues/organisms
       • mRNA expression analysis (DNA arrays and micrarrays) do not always reflect the expression level of proteins
         • merely reflects proteins that are coded for
     • Biological samples (CSF, urine, serum etc) used for proteomic analysis are not suitable for mRNA expression analysis
     • Modifications of proteins that are not apparent from DNA sequencing, i.e. post-translational modifications can be analysed using proteomics
     • Proteomics can be used to analyse the location of proteins
     • Proteomics will ultimately lead to the determination of protein function and therefore, a more detailed understanding of biological systems

2. Methods

   • gel based
     • Protein separation by 2-dimensional gel electrophoresis of intact proteins (DIGE)
       • 2DE
         • developed in the mid to late 1970's
         • separates proteins based on their molecular weight and isoelectric point (pI)
         • can detect proteins using a number of different dyes, including fluorescent ones
         • 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
       • 2DE DIGE
         • 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
   • MS-based "gel-free" methods
     • "shotgun" LC-ESI-MS/MS of total enzymic digest of proteins - peptides
       • Quantification by stable isotope labelling (e.g. ICAT, iTRAQ, SILAC) or label free
     • Protein MS
       • measures m/s (mass/charge ratio)
       • protein/peptide in gas
       • measure intact protein or peptide or 'fragmentation' in mass spectrometer
       • Protein can then be identified based on the ms image produced of teh different amino acids contained within it
     • MALDI-MS
       • Advantages
         • analyte deposited in solid form
         • tolerant to salts
         • produces mostly singly charged ions
       • dissadvantages
         • coupling to peptide fractionation
         • produces mostly singly charged ions
     • Electrospray Ionisation
       • Analyte in aqueous solution sprayed through the bore of a needlie
       • Formation of charged droplets occurs
       • Desolvation of the droplets takes place in a heated capillary
       • Ions are transferred from liquid to gaseous phase by increasing charge
   • Protein Chips/arrays
     • intact proteins
     • e.g. protein, tissue and antibody arrays

3. why perform proteomic analysis (rely on high quality of samples)

   • to provide a mechanistic insight into both the disease pathogenesis and the identification of drug targets
   • Markers:
     • 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.
     • Also performed to find markers for drug development and use

4. proteomic biomarker discovery process

   • Discovery
     • sample accrual
     • proteomics discovery
       • protein separation
       • protein detection
       • Protein characterisation and identification (MS)
     • uses other analytes (anything measurable)
   • Confirmation Assay Development
     • Antibody based
       • Western blotting
       • ELISA
     • Mass Spectrometry based
       • Multiple reaction monitoring (MRM)
     • Multi-analyte assays
   • Validation/Qualification
     • additional clinical samples
     • Large multicentre cohorts
     • Large scale clinical trials
     • "Robust" high-throughput assays
   • Approval & Adoption
     • Regulatory Authorities
     • Clinician Adoption
     • Impact measurement
     • Clinical Assays