Protein-based engineering of the initial acquired enamel pellicle in vivo: Proteomic evaluation

•Rinsing with CaneCPI-5, StN15, hb or combination may increase enamel protection.•Incorporation of proteins/peptide may strengthen the basal layer of the pellicle.•Changes in the precursor proteins of the pellicle may alter the subsequent layers.•Changes in the basal layer of the pellicle may induce long-term changes. To study the proteomic alterations in the initial AEP after rinsing with CaneCPI-5, StN15 or Hb or their combination. In five crossover phases, after prophylaxis, 10 volunteers in 5 consecutive days, rinsed (10 mL, 1 min) with the following solutions: deionized water (H2O- negative control- 1), 0.1 mg/mL CaneCPI-5 (2), 1.88×10−5 M StN15 (3), 1.0 mg/mL Hb (4) or their combination (5). The AEP formed after 3 min was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. Rinsing with the proteins/peptide increased the amounts of proteins in the AEP. The total numbers of proteins identified after rinsing with CaneCPI-5, StN15, Hb or their combination versus water, were 131, 167, 148 and 142, respectively. The treatment with the proteins/peptide or their combination increased proteins that bind calcium, phosphate and interact with distinct proteins, as well as proteins with antimicrobial and acid-resistant properties, such as, Cornifin-B (7.7, 12.6, and 4.3-fold for CaneCPI-5, StN15 and Hb, respectively), isoforms of Cystatin (2.2–2.4-fold for CaneCPI-5 and StN15), Proline-rich-protein 4 (4.3-fold; StN15), Histatin-1 (2.8-fold; StN15) and Hemoglobin (7.7–25-fold for Hb and Combination). Immunoglobulin, Keratin and Histone were exclusively identified upon treatment with the proteins/peptide, alone or combined. Rinsing with proteins/peptide, alone or combined, increased protective proteins in the initial AEP. Our results suggest that rinsing with the proteins/peptide or their combination increases the proteins capable of enhancing the protective function of the basal layer of AEP. [Display omitted]