Standardized Saliva Collection Protocol

Subjects were requested not to eat or drink for at least 30 min before collection. All patients rinsed their mouths with 10 mL of water for 30 s. After 10 min, whole unstimulated saliva collection began. Between 8:00 a.m. and 11:00 a.m., each participant expectorated continuously in a 50 mL sterile propylene tube for a period of 5 min. Additionally, we collected the salivary pellicle deposited on lesions and the contralateral anatomical equivalent of the unaffected oral mucosa using cotton swabs. Immediately after collection, samples were deposited on ice and a protease inhibitor cocktail (cOmplete Tablets EASYpack, Roche)^{26 (link)}. We then centrifuged the samples at 14,000×g at 4 °C for 20 min to eliminate undissolved components and cellular detritus^{27 (link)}. The total protein concentration in the supernatants was determined using the BCA Protein Assay (Thermo Scientific). The samples were stored at − 80 °C for further processing.