Proteomic fingerprints of damage in extracellular matrix assemblies

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.

Peptide location fingerprinting reveals modification-associated biomarkers of ageing in human tissue proteomes

“…The collagen 6 microfibril is a structural ECM assembly comprised predominantly of three alpha chains with alpha-3 being the largest (23). Although previous histological analysis of photoaged skin showed no gross changes to the distribution of the collagen 6 network (24), we recently demonstrated using peptide location fingerprinting that the alpha-3 chain was structurally susceptible to physiological doses of UVR in vitro (10). As these ECM assemblies are markedly long-lived, here we show evidence for the first time that the alpha-3 chain is susceptible to photoageing-dependant modifications in vivo.…”

“…In addition, PSM counts corresponding to each amino acid step size within a protein of one group (buttock) was statistically compared with counts of each corresponding amino acid step size in the other group (forearm) using Bonferroni-corrected, repeated measures paired ANOVA. For further information on peptide location fingerprinting, please refer to previous publications detailing the same process for fibrillin-1 and collagen 6 alpha-3 proteins (10,19).…”

“…In order to address the need for an approach capable of identifying proteins with photoageing specificmodifications, we have developed the publicly available Manchester Peptide Location Fingerprinter (MPLF) webtool which can detect changes in peptide yield along protein structure (10,19). MPLF is an LC-MS/MS proteomic analysis tool which implements peptide location fingerprinting: tryptic peptide spectral counts are mapped to protein regions (specified by the user, or from the UniProt database), relatively quantified per region and statistically tested between groups (Fig.…”

“…Using peptide location fingerprinting, we have previously shown that: i) long-lived macromolecular ECM assemblies exhibit inter-tissue structural diversity (19) and; ii) in vitro UVR-induced damage modifications to protein tertiary/quaternary structures can be detected in cell culture-derived suspensions of isolated ECM assemblies (fibrillin and collagen 6 microfibrils) and their associated receptors (integrins) (10). Here, we show that this same method can be used as a surveying tool capable of detecting statistically significant, photoageing-specific, structure modification-related differences in proteins within human epidermal and dermal proteomes.…”

Complexity of matrix phenotypes

“…Exposure to UVR by broadband UVB and solar-simulated radiation (SSR) causes significant ultrastructural changes exclusively in fibrillin microfibrils as detected by the enhanced periodicity and central bead height of the irradiated microfibrils in vivo and in vitro [ 38 ]. UVR remodels the tertiary and quaternary structure of fibrillin microfibrils making them more susceptible to proteolysis as measured by an increase in liberated peptides as measured via LC-MS/MS analyses following exposure to elastase ( Fig.…”