Proteomic fingerprints of damage in extracellular matrix assemblies

Alexander Eckersley; Matiss Ozols; Ronan O'Cualain; Emma-Jayne Keevill; April Foster; Suzanne Pilkington; David Knight; Christopher E M Griffiths; Rachel E B Watson; Michael J Sherratt

doi:10.1016/j.mbplus.2020.100027

Proteomic fingerprints of damage in extracellular matrix assemblies

Matrix Biol Plus. 2020 Jan 30:5:100027. doi: 10.1016/j.mbplus.2020.100027. eCollection 2020 Feb.

Authors

Affiliations

¹ Division of Cell Matrix Biology & Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.
² Biological Mass Spectrometry Core Research Facility, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.
³ Division of Musculoskeletal & Dermatological Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.
⁴ NIHR Manchester Biomedical Research Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK.

Abstract

In contrast to the dynamic intracellular environment, structural extracellular matrix (ECM) proteins with half-lives measured in decades, are susceptible to accumulating damage. Whilst conventional approaches such as histology, immunohistochemistry and mass spectrometry are able to identify age- and disease-related changes in protein abundance or distribution, these techniques are poorly suited to characterising molecular damage. We have previously shown that mass spectrometry can detect tissue-specific differences in the proteolytic susceptibility of protein regions within fibrillin-1 and collagen VI alpha-3. Here, we present a novel proteomic approach to detect damage-induced "peptide fingerprints" within complex multi-component ECM assemblies (fibrillin and collagen VI microfibrils) following their exposure to ultraviolet radiation (UVR) by broadband UVB or solar simulated radiation (SSR). These assemblies were chosen because, in chronically photoaged skin, fibrillin and collagen VI microfibril architectures are differentially susceptible to UVR. In this study, atomic force microscopy revealed that fibrillin microfibril ultrastructure was significantly altered by UVR exposure whereas the ultrastructure of collagen VI microfibrils was resistant. UVR-induced molecular damage was further characterised by proteolytic peptide generation with elastase followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Peptide mapping revealed that UVR exposure increased regional proteolytic susceptibility within the protein structures of fibrillin-1 and collagen VI alpha-3. This allowed the identification of UVR-induced molecular changes within these two key ECM assemblies. Additionally, similar changes were observed within protein regions of co-purifying, microfibril-associated receptors integrins αv and β1. This study demonstrates that LC-MS/MS mapping of peptides enables the characterisation of molecular post-translational damage (via direct irradiation and radiation-induced oxidative mechanisms) within a complex in vitro model system. This peptide fingerprinting approach reliably allows both the identification of UVR-induced molecular damage in and between proteins and the identification of specific protein domains with increased proteolytic susceptibility as a result of photo-denaturation. This has the potential to serve as a sensitive method of identifying accumulated molecular damage in vivo using conventional mass spectrometry data-sets.

Keywords: AFM, atomic force microscopy; COL6A3, collagen VI alpha 3 chain; Collagen VI microfibril; ECM, extracellular matrix; EGF, epidermal growth factor domain; Fibrillin microfibril; HDF, human dermal fibroblast; LC-MS/MS, liquid chromatography tandem mass spectrometry; Mass spectrometry; PSM, peptide spectrum match; Photodamage; ROS, reactive oxygen species; SSR, solar simulated radiation; TGFβ, transforming growth factor beta; UVR, ultraviolet radiation; Ultraviolet radiation; vWA, von Willebrand factor type A domain.