Services: Protein Profiling – iTRAQ analysis and sample requirements

Protein profiling determines the molar ratios (relative abundance) of identical proteins in compared complex protein mixtures (protein samples). Using iTRAQ analysis, the relative abundance of proteins can be determined in up to eight protein extracts at a time (per analysis). This is achieved by labeling of multi-protein extracts using 8 mass spectrally resolvable, charge-matched labels. iTRAQ labeling is conducted using a commercial kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. Equal amounts (20-100 mg) of total protein from each sample are separately digested with trypsin and labeled with one of iTRAQ reagents (iTRAQ tags are covalently linked to the amino group of the lysine and/or the N-terminal residues of tryptic peptides). The labeled samples are pooled and analyzed by LC/MS/MS for protein identification and quantitation:  identical peptides with different iTRAQ tags are resolved in the same chromatographic peak, the masses of eluted peptides are determined, then, the peptides are fragmented and the fragment masses, as well as, the intensities of the released iTRAQ tags are determined. If losses occur during analysis, each sample experiences the same loss and the ratios (relative abundance) of identical peptides are preserved. The acquired MS data is submitted to database search; the sequence of each analyzed peptide is identified and the relative abundances of identical peptides with different tags are determined. Identified peptides determine the identity and the relative abundance of the corresponding parent proteins.

The following services are provided for iTRAQ analysis:

Service Service Code Sample Requirements
Protein samples are labeled by customers and labeled samples are submitted for LC/MS/MS analysis to the facility.
  • iTRAQ labeling should only be performed using a commercial kit according to the manufacturer's protocol; collected labeled samples are ready for the following LC/MS/MS analysis and can be stored at -20ºC.
Two protein samples are both labeled and analyzed by LC/MS/MS at the facility.
  • Total protein in a sample: 20-100 ug
  • Total protein concentration in a sample: 1-5 mg/ml
  • Sample buffer: 0.5 M TEAB (triethylammonium bicarbonate), pH 8.5, 0.1% SDS
  • Samples should not contain primary amines, thiols (mercaptoethanol, DDT, etc)
Three protein samples are both labeled and analyzed by LC/MS/MS at the facility.
Four protein samples are both labeled and analyzed by LC/MS/MS at the facility.