Use of proteomics methodology to evaluate inflammatory protein expression in tendinitis

In previous studies we established a rat model of acute tendinitis including functional and mechanical measures of healing. Achilles' tendinitis was induced by injection of collagenase, an enzyme that produces localized fiber digestion and edema formation. As quantitative measures of tissue inflammation, hypercellularity and edema were evaluated in injured tendons in comparison with controls. Using the rat tendinitis model, we have applied isotope-coded affinity tag analysis (ICAT) methodology to indicate localized tendon healing by quantitating protein expression. This novel proteomics method allows detection of subtle differences in protein levels that provide a detailed picture of tendinitis healing. The method involves a new class of chemical linkers used to differentially label cysteine residues from similar peptides in control and treated protein samples with heavy (deuterium off of backbone) and light (hydrogen off of backbone) ICAT reagents that are otherwise chemically identical. Proteins were extracted under liquid nitrogen from control untreated or injured Achilles' tendons 72 hours after collagenase-injection. These proteins were digested with endoproteinase Glu-C and trypsin and the resulting peptide mixtures were evaluated using reverse-phase C18 HPLC and Tristricine SDS-polyacrylamide gel electrophoresis. The two ICAT-modified peptide populations were mixed, affinity-purified and analyzed using microcapillary liquid chromatography and electrospray ionization tandem mass-spectroscopy. The process resulted in relative abundance and charge-to-mass ratio data used in conjunction with database searching to identify proteins expressed differentially in the two treatment groups. By analyzing different time periods in the healing process, an accurate model of the healing rat tendon can be made.