Proteomic analysis of tendons at different risk of injury.

Tendon injury is a common problem in both human and veterinary medicine, which impacts on the both the quality of life of the individual in question but also a financial implication on the supporting health system. Previous research into the pathogenesis of tendon injury and also the healing and treatment options available are limited. This one year project aimed to advance our knowledge in proteomics of human and equine tendons by developing proteomic methodologies for the analysis of the normal protein composition. In order to build a proteomic tendon library three different protein extraction protocols were considered, urea, guanidine and Rapigest. Results revealed urea had a poor extraction ability and that guanidine and Rapigest had a better success rate. Further work to combine these to agents would be worthwhile in the future. Further to this to this analysing spatial distribution of these proteins was then investigated in equine tissue. This work made some progress in optimizing a protocol, finding trypsin digestion worked better when incubated for 16 hours and also when the digestion occurred with an ammonium bicarbonate buffer. The fourth chapter investigated whether it is possible to detect in vivo changes in the proteomic composition of dialysis fluid subsequent to shockwave therapy of human Achilles tendon. This study revealed that current methods were sensitive enough to ascertain the protein composition but further work is needed to ascertain what significant protein changes occur in response to therapy. Finally tendon protein turnover was explored in an in-vivo model. Results confirmed proteins within the non collagenous matrix had a higher rate of protein turnover compared to components of the collagenous matrix. All four projects have been the first of their kind so optimisiation and a ‘look and see’ approach was required, which has laid foundations for several subsequent studies