Rational selection of peptide epitope templates for proteins imprinting

Background The main disadvantage in the use of proteins as templates in protein imprinting is their 3D-structure, complex and flexible, that unfolds easily. Decreasing the complexity of the protein template seems the best strategy, as demostrated by “epitope imprinting”. Yet, in all current protein MIP approaches, internal epitopes were not accessible and thus, have never been considered as templates, bringing to the loss of a huge number of potential epitope-candidates. Furthermore, in many cases conserved epitopes of diagnostic interest are located at the core of the protein, where these are not exposed to the pressure of the immune system. Objective Here we propose a rational method for the selection of epitopes for protein imprinting that enables targeting all epitopes, either exposed or internal. Named fingerprint imprinted polymers (FIP), in analogy with the fingerprinting analysis (the use of the constituent peptides to identify a protein). Methods The FIP method is based on the following steps for identifying a peptide candidate for imprinting: (I) in silico cleavage of the protein sequence by various cleaving agents; (II) selection of the resulting peptides based on length and hydrophylicity ; (III) screening of each peptide candidate against the entire protein sequences databank (e.g. UniProtKB) to eliminate candidate sequences that are not unique enough. A probability score of uniqueness is associated to the each peptide candidate, allowing the selection of unique sequences as templates. Results To proof the principle, NT-proBNP maker of cardiovascular risk, was chosen. The in silico analysis of NT-proBNP sequence allowed to individuate two peptide candidates, next used as templates for the preparation of NT-pro-BNP specific FIPs and tested for their ability to bind the NT-proBNP peptides in complex samples. Results indicated remarkable imprinting factor (IF 10), binding capacity of 0.5-2 mg/g, in line with many affinity materials, ability to rebind the 40% of template in a complex sample, composed of the whole digests of NT-proBNP. Conclusions Results supported the validity of the method here proposed. Key point is the fragmentation of the protein into peptides. The advantage is the ability to circumvent the issue of the folding of the protein