Optimizing Collection of Acquired Pellicle in Vitro for Pproteomic Analysis

Objectives: This study compared the proteomic profile of the acquired enamel pellicle (AEP) formed in vitro using different collection solutions.
Methods: Four hundred thirty-two bovine enamel specimens (4X4 mm) were prepared and divided into four groups. Non-stimulated saliva was provided by nine subjects and pooled. The AEP was formed on the enamel specimens by immersion in the pooled saliva (250 µL) for 120 min at 37°C under agitation. Saliva was changed every 30 min. After formation, AEP was collected with wick filter papers embedded in four different solutions: 1) 3% citric acid (CA); 2) 0.5% sodium dodecyl sulfate (SDS); 3) CA followed by SDS (CA+SDS); 4) SDS followed by CA (SDS+CA). The AEPs collected were processed for analysis by LC-ESI-MS/MS.
Results: The total amount of AEP proteins recovered was quite similar for all the groups, ranging between 26 and 33 µg. The total numbers of proteins identified in each group were 40, 21, 28 and 41 for the groups CA, SDS, CA+SDS and SDS+CA, respectively. Among them, 15, 14, 14 and 9 are proteins typically found in the AEP. The respective figures for proteins found exclusively in one of the groups was 8, 0, 5 and 4, respectively. Fifteen proteins were identified in all the groups, most of which are proteins typically described in the AEP, such as Pancreatic alpha-amylase, Submaxillary gland androgen-regulated protein 3B, Immunoglobulin heavy constant alpha 1, Immunoglobulin heavy constant alpha 2, two isoforms of Alpha-amylase, three isoforms of Cystatin, Lysozyme C and Statherin. Remarkably, mucin was only found in CA and CA+SDS groups.
Conclusions: Considering practicality, quantity and quality of the identified proteins, CA seems to be the best solution to be used for collection of AEP proteins.