556 Advances in extracellular matrix proteomics through optimised sample preparation

Fibrillin-rich microfibrils (FRM) exist both as independent tissue components: (i) in the eye and the skin (papillary dermis) and; (ii) in association with elastic fibres (reticular dermis). Their loss from the papillary dermis acts as an early biomarker of UV-mediated tissue remodelling. We have shown previously that FRM ultrastructure is sensitive to solar simulated radiation. However, it is not known if UV radiation also affects FRM protein composition or fibrillin-1 structure. The proteomic analysis of many extracellular matrix assemblies is problematic as their tightly folded structure can mask cleavage sites. In this study we aimed to develop new specimen preparation methods to characterise the biochemistry of FRMs using mass spectrometry (MS). We extracted and isolated FRMs from human ocular tissue (n=3; 55-77 years) by collagenase IA digestion and size exclusion chromatography. Three methods of pre-MS sample preparation were compared: i) solubilisation followed by trypsin digestion; ii) solubilisation followed by elastase digestion and; iii) high temperature ThermoScientific™ SMART Digestion™ without prior solubilisation. Samples were run on an Orbitrap™ Elite™ LC-MS. Compared to conventional trypsin digestion: i) elastase digestion increased the 1o amino acid coverage of fibrillin-1 (8% to 38%) and; ii) SMART Digestion™ enhanced the detection of FRM-associated proteins (from 3 to 9). Thus, these novel digestion methods successfully enhance our ability to characterise the biochemistry of an important dermal supra-molecular assembly. We are currently applying these techniques in combination with atomic force microscopy to characterise the biochemistry and ultrastructure of ocular derived-, skin derived- and cutaneous fibroblast-derived FRMs.