1. Isobaric Tagging – Isobaric tagging is based upon chemical isotope incorporation with tags of identical chemical structure and the same total mass. The labile portion of the tag that is released during fragmentation (i.e. reporter ions) will vary in mass and relative quantitation is based on the intensity of these reporter ions. Isobaric tagging approaches enable multiplexing. iTRAQ, Tandem Mass Tag (TMT), ICAT

2. Metabolic Labeling – Stable isotope labeling using amino acids in cell culture (SILAC) involved incorporation of isotopes through the use of media containing 13C or 15N labeled amino acids. Relative quantitation is based on the intensities of the light and heavy labeled peptides. (Please note that we do not provide cell culture services but can support experimental design and proteomic analysis of SILAC labeled samples). SILAC

3. Label-free – Method aims to determine the relative amount of proteins in two or more biological samples. Unlike other methods for protein quantitation, label-free quantification does not use a stable isotope containing compound to chemically bind to and thus label the protein. Label-free quantification may be based on precursor signal intensity or on spectral counting. Spectral Counting is based upon the assumption that the number of MS/MS spectra identified for a given protein is directly proportional to the abundance of that protein in the sample. Spectral Counting can be used to obtain relative quantitation of proteins in a complex sample without the requirement of labeling.

Please note:
1. For these experiments we recommend triplicate analysis of each biological replicate, to increase statistical significance of the peptide (and thus protein) fold-changes measured. This type of experiment can be extended to studies including protein expression as a function of time given an external stimuli or gene knockout, for instance. Label-free differential expression is only available on separations utilizing a single dimension of LC prior to MS/MS.

2.Both Discovery and Targeted experiments are time and labour intensive. The success of these approaches relies on detailed planning and choosing the appropriate sample preparation techniques