Proteomics of tendon ECM assembly

  The ability of tendon to transduce tensile force to the skeleton relies on 10s of 1000s of long (>100 microns) extracellular collagen fibrils that are in parallel register with the long axis of the tendon. Electron microscopy studies of developing mouse‐tail tendon show that at E13.5, the tissue is predominantly composed of cells with little or no matrix. By E15.5, intracellular fibrils can be seen. The long collagen fibrils are deposited one‐by‐one by ‘finger‐like’ projections of the plasma membrane into hexagonally packed bundles of the fibrils. Moreover, the fibrils have a precise interfibrillar spacing of approximately 30 nm. The aim of this work was to identify proteins whose patterns of expression change during tail development in order to understand better the molecular basis of intracellular collagen fibril assembly, tubule trafficking and supramolecular organization of the fibrils in the embryonic mouse tendon. Materials and methods  Fresh embryonic tail tissue was harvested and proteins extracted into buffer containing urea, thiourea, detergent and DTT (DL‐dithiothreitol). Proteins (approximately 100 µg) were first separated by isoelectrofocusing on 24 cm, linear immobilized pH gradient strips (pH 4–7). Strips were reduced with 1% DTT and alkylated with 5% iodoacetamide before second dimension separation by SDS‐PAGE on an 11% gel. Gels were stained with either 0.1% colloidal Coomassie Blue or silver nitrate. Coomassie‐stained gels were used for MS analysis. Protein spots of interest were excised from the 2D gel, destained, reduced, alkylated and digested overnight with trypsin. The resulting peptides were then extracted and analysed by LC‐MS/MS. The data were used to search against SWISSPROT and Tremblnew public protein databases. Results  We have established reproducible extraction conditions and separation methods to obtain 2D gels containing >500 protein ‘spots’ that are visible by Coomassie Blue staining. Discussion  Our approach is to extract proteins from whole embryonic tail from both E13.5 and E15.5 tissues, separate the proteins by 2D‐gel electrophoresis, highlight key changes in the spot profile and identify these proteins by trypsin digestion, q‐TOF mass spectrometry and database matching. A surprising observation was the abundance of intracellular proteins involved in protein folding and trafficking. A poster will be presented that lists the first proteins that we have identified.