The discovery phase of proteomics projects is aimed at finding proteins which are differentially expressed under differing growth or health conditions. Biomarkers or other target proteins are identified by comparative proteomics.
Different strategies can be applied with regard to their strengths and customer's preferences:
- two-dimensional gel electrophoresis (2-DE) using either regular silver staining or differential fluoroescent labelling
- mass spectrometry-based approaches with differential isobaric labelling like iTRAQ and TMT and multiplexed analysis of samples.
- label-free quantification of samples after proteolysis and LCMS/MS of each sample
Two-dimensional gel electrophoresis features the following properties:
- separation on intact protein level, and thus
- resolution of PTM variants and other species of the same protein
- separation of hundreds or thousands of proteins and protein species
- more visual result
- only significantly regulated proteins are subsequently identified by MS/MS
- limited suitability for highly hydrophobic or very large proteins
LCMS-based techniques like iTRAQ and TMT offer
- large scale identification and quantification of all detectable proteins
- less biased with regard to chemicophysical protein properties
TMT is a trademark of Proteome Sciences plc.