Jeremy Horst Keeper ^1* Jong Seto ^1,2,3* Ersin Emre Oren ⁴ Orapin Veerayutthwillai Horst ^1* Ling-Hong Hung ⁵ Ram Samudrala ⁶

• ¹ University of California, San Francisco, San Francisco, United States
• ² Arizona State University, Tempe, Arizona, United States
• ³ The Molecular Foundry, Berkeley Lab (DOE), Berkeley, California, United States
• ⁴ TOBB University of Economics and Technology, Ankara, Ankara, Türkiye
• ⁵ University of Washington Tacoma, Tacoma, Washington, United States
• ⁶ University at Buffalo, Buffalo, New York, United States

Extracellular matrices direct the formation of mineral constituents into self-assembled mineralized tissues. We investigate the protein and mineral constituents to better understand the underlying mechanisms that lead to mineralized tissue formation. Specifically, we study the protein-hydroxyapatite interactions that govern the development and homeostasis of teeth and bone in the oral cavity. Characterization would enable improvements in the design of peptides to regenerate mineralized tissues and control attachments such as ligaments and dental plaque. Progress has been limited because no available methods produce robust data for assessing organic-mineral interfaces. We show that tooth enamel pellicle peptides contain subtle sequence similarities that encode hydroxyapatite binding mechanisms, by segregating pellicle peptides from control sequences using our previously developed substitution matrix-based peptide comparison protocol with improvements. Sampling diverse matrices, adding biological control sequences, and optimizing matrix refinement algorithms improves discrimination from 0.81 to 0.99 AUC in leave-one-out experiments. Other contemporary methods fail on this problem. We find hydroxyapatite interaction sequence patterns by applying the resulting selected refined matrix ("pellitrix") to cluster the peptides and build subgroup alignments. We identify putative hydroxyapatite maturation domains by application to enamel biomineralization proteins and prioritize putative novel pellicle peptides identified by In stageTip (iST) mass spectrometry. The sequence comparison protocol outperforms other contemporary options for this small and heterogeneous group and is generalized for application to any group of peptides. As a result, this platform has broad impacts on peptide design with direct applications to microbiology, biomaterial design, and tissue engineering.