Samdi mass spectrometry for high yield protein modification reaction development

Efficient chemical strategies that attach synthetic molecules to desired positions on protein surfaces are useful tools in the field of chemical biology and represent one major prerequisite for the development of new drugs and materials. Protein modification with polyethylene glycol (PEG) groups is indeed routinely performed on therapeutic proteins to improve serum half-life, or even cytotoxins or imaging agents are efficiently conjugated to cancer-targeting elements. In a typical approach, a synthetic functional group of interest is attached to a uniquely reactive amino acid group introduced by recombinant methods. Most bioconjugation reactions, however, do not reach full conversion. Therefore the development of a straightforward and reliable method to increase the extent of conversion into bioconjugates would be very helpful. In this perspective, we developed a generalizable combinatorial peptide library screening platform suitable for the identification of sequences displaying high levels of reactivity toward a desired bioconjugation reaction. This was achieved by using SAMDI MS technique (Self-Assembled Monolayer and Desorption/Ionization Mass Spectrometry) as a new, efficient and simple method for the evaluation of highly reactive amino acid motifs. The bioconjugation reaction we selected is the oxidative modification of electron-rich tyrosine residues performed using cerium(IV) ammonium nitrate (CAN) as oxidant reagent. The peptides were identified on a 361-member hexapeptide array, wherein the two N- and C-terminal residues to the target residue were varied. The arrays were prepared by immobilizing the peptides to a self-assembled monolayer of alkanethiolates on gold and could therefore be analyzed by mass spectrometry. We found that the most reactive peptides had either a serine N-terminal to the tyrosine residue or another tyrosine in proximity of the reactive site. Conversely, peptides displaying the lowest conversion level contained a positive charged residue: histidine, lysine or arginine, where the lowest relative activity was reached with arginine and leucine as C- and N- terminal residues, respectively. This study provides an important example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.