Proteonomics

Proteonomics

1. >10,000 proteins expressed at any one time

   • Traditional Techniques e.g. Western blotting, or microscopy
     • Visualise individual proteins by detecting with an antibody
       • 1-5 proteins can be studied in a single sample
   • Proteonomics e.g. mass spectrometry
     • Measure proteins directly
       • 1000s of proteins in a single sample

2. Mass Spectrometry: Sample Preparation

   • 1. Complex protein mixture is separated using Sodium Dodcyl Sulphate Polyacryl Gel Electrophoresis (SDS-PAGE). Cut out bands to give simplfied proteins mixtures
     • 2. Tryptic digest to produce peptides. Trypsin is a proteolytic enzyme and cuts after lysine and arginine amino acids. 6-20 amino acids long (small enough for mass spec but long enough to identify proteins)
       • 3. Peptide separation. High Pressure Liquid Chormatography (HPLC) separates peptides according to thier hyrdrophobicity
         • 4. Electrospry ionisation of peptides. Electrospray ionisation converts the liquid sample to a charged, gaseous phase which is suitable for injection into the mass spectrometer
   • Liquid chromatography coupled mass spectrometry (LC-MS). mass/charge ratio is measured and the protein can be identified
   • Data analysis is done using - 'Xcalibur' which produces a mass spectrum, 'Maxquant' which can perform peptide and protein searches

3. Quantitative mass spectrometry

   • 1. Relative quatification e.g. there is 3 times as much protein X in sample 1 than sample 2. Techniques include SILAC, iTRAQ
     • SILAC - Stable isotope labeling of amino acids in culture
   • 2. Abosolute quatification e.g. there are 3 femtomoles of protein Z in sample 1 and 6 femtomoles in sample 2. Techniques include AQUA, QconCAT
     • AQUA; Absolute Quantification, enzymatic digest with trypsin, add 1fM of protein X and do LC-MS
   • AQUA v SILAC

Bilder und Dateien der Mindmap