Fibrillin microfibrils are extensible polymers that endow connective tissues with long-range elasticity and have widespread distributions in both elastic and non-elastic tissues. They act as a template for elastin deposition during elastic fibre formation and are essential for maintaining the integrity of tissues such as blood vessels, lung, skin and ocular ligaments. A reduction in fibrillin is seen in tissues in vascular ageing, chronic obstructive pulmonary disease, skin ageing and UV induced skin damage, and age-related vision deterioration. Most mutations in fibrillin cause Marfan syndrome, a genetic disease characterised by overgrowth of the long bones and other skeletal abnormalities with cardiovascular and eye defects. However, mutations in fibrillin and fibrillin-binding proteins can also cause short-stature pathologies. All of these diseases have been linked to dysregulated growth factor signalling which forms a major functional role for fibrillin.

Elastic fibers comprising fibrillin microfibrils and elastin are present in many tissues, including the skin, lungs, and arteries, where they confer elasticity and resilience. Although fibrillin microfibrils play distinct and tissue-specific functional roles, it is unclear whether their ultrastructure and composition differ between elastin-rich (skin) and elastin-poor (ciliary body and zonule) organs or after in vitro synthesis by cultured cells. Here, we used atomic force microscopy, which revealed that the bead morphology of fibrillin microfibrils isolated from the human eye differs from those isolated from the skin. Using newly developed pre-MS preparation methods and LC-MS/MS, we detected tissue-specific regions of the fibrillin-1 primary structure that were differentially susceptible to proteolytic extraction. Comparing tissue- and culture-derived microfibrils, we found that dermis- and dermal fibroblast–derived fibrillin microfibrils differ in both bead morphology and periodicity and also exhibit regional differences in fibrillin-1 proteolytic susceptibility. In contrast, collagen VI microfibrils from the same dermal or fibroblast samples were invariant in ultrastructure (periodicity) and protease susceptibility. Finally, we observed that skin- and eye-derived microfibril suspensions were enriched in elastic fiber– and basement membrane–associated proteins, respectively. LC-MS/MS also identified proteins (such as calreticulin and protein-disulfide isomerase) that are potentially fundamental to fibrillin microfibril biology, regardless of their tissue source. Fibrillin microfibrils synthesized in cell culture lacked some of these key proteins (MFAP2 and -4 and fibrillin-2). These results showcase the structural diversity of these key extracellular matrix assemblies, which may relate to their distinct roles in the tissues where they reside.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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.

No abstract

No abstract

No abstract

In ageing tissues, long-lived extracellular matrix (ECM) proteins are susceptible to the accumulation of structural damage due to diverse mechanisms including glycation, oxidation and protease cleavage. Peptide location fingerprinting (PLF) is a new mass spectrometry (MS) analysis technique capable of identifying proteins exhibiting structural differences in complex proteomes. PLF applied to published young and aged intervertebral disc (IVD) MS datasets (posterior, lateral and anterior regions of the annulus fibrosus) identified 268 proteins with age-associated structural differences. For several ECM assemblies (collagens I, II and V and aggrecan), these differences were markedly conserved between degeneration-prone (posterior and lateral) and -resistant (anterior) regions. Significant differences in peptide yields, observed within collagen I α2, collagen II α1 and collagen V α1, were located within their triple-helical regions and/or cleaved C-terminal propeptides, indicating potential accumulation of damage and impaired maintenance. Several proteins (collagen V α1, collagen II α1 and aggrecan) also exhibited tissue region (lateral)-specific differences in structure between aged and young samples, suggesting that some ageing mechanisms may act locally within tissues. This study not only reveals possible age-associated differences in ECM protein structures which are tissue-region specific, but also highlights the ability of PLF as a proteomic tool to aid in biomarker discovery.