Proteomics as a Tool to Gain Next Level Insights into Biopolymer Modifications

The distribution of introduced photo-crosslinkable moieties onto a protein can affect a biomaterial’s crosslinking behavior, and therefore also the mechanical and biological properties. However, this information is also vital when developing biomaterials for tissue engineering and regenerative medicine applications. In the present work, photo-crosslinkable moieties have been introduced on the primary amine groups of: (i) a recombinant collagen peptide (RCPhC1) with a known amino acid (AA) sequence, and (ii) bovine skin collagen (COL BS) with an unknown AA sequence. The degree of substitution (DS) was quantified with two standard techniques: an ortho-phthalic dialdehyde (OPA) assay and 1H-NMR spectroscopy. However, neither of both provides information on the type and exact location of the modified AAs. Therefore, for the first time, proteomic analysis was evaluated herein as a tool to identify functionalized AAs as well as the exact position of the photo-crosslinkable moieties along the AA sequence, thereby enabling an in-depth, unprecedented characterization of functionalized biopolymers. Moreover, our strategy also enabled to visualize the spatial distribution of the modifications within the overall structure of the protein. Proteomics has proven to provide unprecedented insight in the distribution of photo-crosslinkable moieties along the protein backbone, undoubtedly contributing to superior biomaterial design in the future.