In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials

BackgroundElastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown.MethodsSmall peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry.ResultsMS2 data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations.ConclusionsThe study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials.General significanceThe study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin.

► In vitro cross-linking of short elastin peptides yields elastin-like biomaterials.
► Software was developed to allow identification of native polyfunctional cross-links.
► Intra- and intermolecular cross-links characteristic of elastin were identified.
► In vitro formation of desmosine was proven by means of mass spectrometry.