Abstract

Quantification of gene expression is the sine qua non in understanding how the components of a biological cell or an organism change and interact following external or internal perturbations. Analytical techniques that combine stable isotopic protein and peptides labelling and mass spectrometric analysis are becoming increasingly popular in determining protein abundance. The common motif, which recurs in these bio-analytical methods, is the parallel labelling of the protein samples with stable isotopes. The ratio of the differently labelled peptide ions observed in the mass spectrum reflects the differences in concentration between the species in the combined mixture. Isotopes can be introduced using labelled nutrients prior to proteolytic digestion or by chemical derivatisation of the peptide functional groups after proteolysis. Although metabolic incorporation of labelled isotopes provides a more global quantification strategy, chemical derivatisation based approaches are also suitable for those laboratories with basic proteomics facilities. Advantages and limitations of these techniques are reviewed, highlighting examples of specific biological applications.

Keywords: MALDI matrix-assisted laser desorption ionisation, ES electrospray, RP-HPLC reversed phase high performance liquid chromatography, SDS PAGE sodium-dodecyl sulphate polyacrylamide electrophoresis, CAD collisionally activated dissociation, PMF peptide mass fingerprinting, ICAT Isotope coded affinity tag, GLaD guanidino-labelling derivatisation