There are multiple options for quantitative analysis of proteins and peptides, each with their own advantages and disadvantages. We encourage you to contact us directly to discuss the specifics of your project so that we can help you to determine the best approach.

  Label free (DDA and/or DIA) Isobaric labelling (iTRAQ and TMT)        Metabolic Labeling       
Cell Lines
Tissues
Serum / Plasma - -
Labeling Technique - In vitro  In vivo 
No. of Samples that can be compared Unlimited
(Samples analyzed individually)

4 or 8 (iTRAQ);
6 or 11 (TMT)

3 or 6

 

Discovery Proteomics

  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 either by iTRAQ and/or Tandem Mass Tag (TMT) 

  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 experiments).

  3. Label-free
    The 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.

Please note:

  • For these experiments we recommend triplicate analysis of each biological replicate, to increase the 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.

  • 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.