Abstract
Introduction
Proteomic analysis has the ability to reveal both the different types and abundances of proteins in a sample. To date, proteomic analysis has received limited attention in the field of tendon research, with mainly ex vivo investigations being undertaken to characterize the tendon proteome. A significant development would be the ability to detect in vivo changes in the proteomic composition as this could have clearer and more direct understanding on the efficiency of therapies. It is well documented that sample preparation is one of the most crucial steps in obtaining high quality resolution of proteins in mass spectrometry. Biological samples can vary in complexity, and minimization of this through sample handling and cleaning can drastically improve the resolved peptide spectra. During this investigation, samples of microdialysis media from the peritendinous space of the Achilles tendon pre or post shockwave therapy were used to establish whether the in vivo identification and quantification of proteins was possible.
Materials and Methods
Six microdialysis samples were obtained from human subjects before (controls) or after shock wave therapy on their achilles tendon. Samples were concentrated and intefering substances removed using StrataClean™ resin. Reduction, alkylation and an In-solution tryptic digestion was performed with the prior addition of 1% Rapigest SF solution. Samples were then analysed by Liquid Chromatography Mass Spectrometry/Mass Spectrometry. Data files were searched using IPI-human database using Mascot Search Engine. Relative quantification was performed between groups by ProgenesisQI.
Results
An increase in protein concentration within dialysis media following treatment was observed. Protein identifications included extracellular matrix proteins such as collagens and proteoglycans, and cellular proteins. In total there were 93 quantifiable proteins identified, of these 80 proteins had a p<0.05 and 2 fold change difference; 61 higher and 19 lower in post shock wave therapy.
Discussion
We are able to identify a range of differentially expressed proteins in dialysis fluid subsequent to shock-wave therapy of the Achilles tendon. This type of analysis could be used to monitor efficiencies, and identify mechanisms of therapies by detection of specific biomarkers.
