Mass Spectrometry-Based Proteomic Approaches for Salivary Biomarkers in Patients with Fixed Orthodontic Appliances and the Invisalign System: A Non-Randomized Clinical Trial

Background/Objectives: Early detection of biological changes is crucial for effective monitoring of orthodontic tooth movement (OTM). Research that elucidates the connection between OTM and its proteomic profile remains limited. This study aimed to identify salivary protein biomarkers specific to fixed orthodontic appliances (FOA) and Invisalign (IN) treatment modalities. Methods: Unstimulated saliva was collected from 17 patients treated with FOA and 6 patients treated with IN before treatment (T0), and at 3 (T1) and 6 (T2) months after therapy initiation. Salivary proteomes were analyzed using liquid chromatography-tandem mass spectrometry, followed by label-free protein quantification and identification with MaxQuant. Heatmap visualization, Partial Least Squares Discriminant Analysis (PLS-DA), and ANOVA were performed using MetaboAnalyst (P≤0.05) to identify differentially expressed proteins (DEPs). Gene Ontology enrichment analysis and Protein-Protein Interaction (PPI) network analysis were conducted on the identified DEPs. Results: PLS-DA demonstrated clear separation between T0, T1, and T2 in both groups. From 17,603 DEPs, 12 and 50 top proteins were identified between FOA and IN at T1 and T2, respectively. Three DEPs—CD19, FAM20C, and GNRHR—were identified in both groups. Their expression levels significantly decreased (P<0.05) at T1 and T2 within each group, with no significant differences in CD19 and FAM20C between the two groups at T0, T1, and T2. PPI revealed associations between these proteins and OTM-related proteins, including fibronectin, RANK, osteopontin, dentin sialophosphoprotein, SOX9, alkaline phosphatase, collagen alpha-1, and collagen alpha-2.Conclusions: CD19, FAM20C, and GNRHR are involved in OTM, suggesting their potential utility as salivary biomarkers.