Services: Protein Profiling – cICAT analysis and sample requirements

Protein profiling determines the molar ratios (relative abundance) of identical proteins in compared complex protein mixtures (protein samples). Using cICAT analysis, the relative abundance of proteins can be determined in two protein extracts at a time (per analysis). This is achieved by labeling of multi-protein extracts using two mass spectrally resolvable, charge-matched labels. cICAT labeling is conducted using a commercial kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. Equal amounts (100 mg) of total protein from each sample are separately labeled with one of cICAT reagents (cICAT tags are covalently linked to the thiol group of cysteine residues). The labeled samples are pooled and digested by trypsin. Labeled tryptic peptides (containing cysteine residue) are separated from the rest of tryptic digest and analyzed by LC/MS/MS for protein identification and quantitation:  identical peptides with different cICAT tags are resolved in the same chromatographic peak, the masses and the intensities of eluted peptides are determined, then, the peptides are fragmented and the corresponding fragment masses 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.

Two different services are provided for cICAT 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.
  • cICAT 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.
Protein samples are both labeled and analyzed by LC/MS/MS at the facility.
  • Total protein in a sample: 100 ug
  • Total protein concentration in a sample: ≥ 1.3 mg/ml
  • Sample buffer: 50 mM Tris-HCl, pH 8.5, 0.1% SDS
  • Samples should not contain thiols (DDT, mercaptoethanol, etc.)