Optimizing the formation of the acquired enamel pellicle in vitro for proteomic analysis

Abstract Saliva is the major contributor for the protein composition of the acquired enamel pellicle (AEP), a bacteria-free organic layer formed by the selective adsorption of salivary proteins on the surface of the enamel. However, the amount of proteins that can be recovered is even smaller under in vitro condition, due to the absence of continuous salivary flow. Objective This study developed an in vitro AEP protocol for proteomics analysis using a new formation technique with different collection solutions. Methodology 432 bovine enamel specimens were prepared (4x4 mm) and divided into four groups (n=108). Unstimulated saliva was provided by nine subjects. The new AEP formation technique was based on saliva resupply by a new one every 30 min within 120 minutes at 37ºC under agitation. AEP was collected using an electrode filter paper soaked in the collection solutions according with the group: 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 pellicles collected were processed for analysis through LC-ESI-MS/MS technique. Results A total of 55 proteins were identified. 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. Twenty-three typical AEP proteins were identified in all groups, but Mucin was only found in CA and CA+SDS, while three types of PRP were not found in the SDS group. Moreover, a typical enamel protein, Enamelin, was identified in the CA+SDS group only. Conclusion The new technique of the in vitro AEP formation through saliva replacement was essential for a higher number of the proteins identified. In addition, considering practicality, quantity and quality of identified proteins, citric acid seems to be the best solution to be used for collection of AEP proteins.


Introduction
Saliva is formed mainly by the secretion of salivary glands. This fluid is essential for the homeostasis of the oral cavity, since it cleans, lubricates and protects the oral tissues, as well as acting as a buffering agent and source of calcium and phosphate ions for remineralization of the teeth. 1 Moreover, saliva is the major contributor for the protein composition of the acquired enamel pellicle (AEP), a bacteria-free organic layer formed by the selective adsorption of salivary proteins on the surface of the enamel, 2 but containing also carbohydrates, neutral lipids, phospholipids and glycolipids. [3][4][5] These organic components grant important functions to the AEP that acts as a diffusion barrier, reducing the direct contact of the acids with the tooth surface, slowing down tooth dissolution. 1,6,7 The ability of the AEP to protect the enamel surface against acids is due mainly to its protein composition, especially by the proteins present in the basal layer.
These remain in the AEP after exposure to acids 8 and are currently objects of great interest, since they might protect against dental caries and erosion. In the last few years, proteomic approaches have been used to identify these proteins [9][10][11][12] so that they can be added to dental products which, when applied, could modify the composition of the AEP, increasing its protective potential against acids. 13 One of the main difficulties faced in the studies involving proteomic analysis of the AEP is the small amount of proteins that can be obtained, which can impair analysis, both in in vitro, in situ and in vivo.
The amount of proteins that can be recovered is even smaller under in vitro condition, due to the absence of continuous salivary flow. Moreover, in the in vivo studies available so far, AEP samples collected from 8-10 volunteers are pooled in order to obtain enough proteins to be analyzed by mass spectrometry, [10][11][12][14][15][16][17][18] which does not allow proper assessment of the biological variation of the samples. In these in vivo studies, the collection of the AEP samples is done with filter paper soaked in 3% citric acid.
Recently, the proteome of the acquired pellicle formed in situ on ceramic specimens and collected by incubation in Tris-HCl buffer containing Triton X-100 followed by ultrasonication in RIPA buffer was analyzed from individual volunteers, with high inter-individual and inter-day consistency. 19 However, the protocol of collection of the AEP employed by Delius 2017 19 is not viable to be employed in vivo, since Triton X-100 is toxic and sonication is not possible. In addition, 0.5% dodecyl sodium sulphate (SDS) has been employed for the collection of AEP samples for analysis of individual proteins by immunoblotting, 20 but SDS was not tested for collection of AEP samples for proteomic analyses yet.
Thus, the aim of this study was to develop an in vitro AEP formation protocol comparing different collection solutions for shotgun proteomic analysis.
The solutions tested (3% citric acid and 0.5% SDS, alone or in combination) were chosen based on their potential to be employed under in vivo conditions, which would allow individual analysis and better assessment of biological variation among the volunteers in future studies.

Formation of AEP in vitro
For the formation of the AEP, the specimens were placed in 96-well microplates in which 250 µL of saliva were added. The AEP was then allowed to form for 120 min. For the constant control of the temperature and agitation, a ThermoMixer ® (Eppendorf ThermoMixer ® C, Hamburg, Germany) was used at 37°C, under agitation. The mainly particularity in this study was the new methodology adopted regarding the resupply of saliva. For this, during the AEP formation (120 min), saliva was exchanged three times (every 30 min).
This way, the previous saliva was removed and a new sample was immediately added (250 µL).

Collection of the AEP
After the formation of AEP, the specimens were immediately withdrawn from saliva and washed with a small spray of deionized water for three seconds and air dried. The AEP was collected using an electrode filter paper 5 × 10 mm (Electrode Wick, Bio-Rad, Hercules, CA, USA) soaked in the collection solutions according with the respective group. The excess of the acid was removed with absorbent paper. For CA+SDS and SDS+CA groups, one filter paper was used for the first solution and a new filter paper was used for the second one. One filter paper was used for 6 specimens only and then resupplied by a new one.
For AEP collection, each paper soaked with their respective solution was rubbed (no pressure) on the enamel surface, with the aid of tweezers. 16 The filter papers used to collect AEP from the specimens of the same group were placed in 2 mL tubes and stored at -80°C. The experiment was repeated for additional 2 consecutive days.

Shotgun proteomics analysis by NanoLC-ESI-MS/MS
The methods were exactly as described elsewhere. 17 The papers with the samples were cut into small pieces with the aid of sterile scissors and tweezers. The filter papers containing the AEP collected from 3 different days (triplicate collection) for each of the groups were pooled to obtain enough amount of AEP proteins to be submitted to the proteomic analysis.  Table   S1).

Results
The total amount of AEP proteins recovered was very similar for all the groups, ranging between 26 and 33 µg. A total of 55 proteins were identified ( Figure 1), among which are 20 proteins typically found in the AEP, such as two isoforms of Alpha-amylase, two isoforms of Basic salivary proline-rich protein, three isoforms of Cystatin, five isoforms of Hemoglobin, Lysozyme, Mucin-7, Pancreatic alpha-amylase, Proline-rich protein 4, Protein S100-A9, Salivary acidic proline-rich phosphoprotein ½, Statherin and Submaxillary gland androgen-regulated protein 3B (Table S1).
The total numbers of proteins identified in each group were 40, 21, 28 and 41 for CA, SDS, CA+SDS and SDS+CA, respectively. Among them, 15, 14, 14 and 9 are proteins typically found in the AEP ( Table   1). Additionally, the proteins found exclusively in one of the groups was 8, 0, 5 e 4 for the groups CA, SDS, CA+SDS and SDS+CA, respectively (Table 1; Figure 1).  (Table S1).
Remarkably, Mucin-7 was only identified in the CA and CA+SDS groups, while Protein S100-A9 was only found in the CA and SDS+CA groups. On the other hand, isoforms of Hemoglobin were only detected in the SDS and SDS+CA groups. Moreover, a typical enamel protein, Enamelin, was identified in the CA+SDS group only. Furthermore, 3 types of PRP were not found in the SDS group (Table S1).

Discussion
The proteomic analysis of AEP formed in vitro is an important tool in pre-clinical studies since it allows preliminary evaluation of preventive agents for dental caries and dental erosion. In addition, in in vitro studies it is possible to recover the enamel specimens over which the AEP is formed to be submitted to distinct tests, which is not feasible in vivo. However, to date there is only one study where the proteomic To date, most of the studies available in the literature employ 3% citric acid for collected of the acquired pellicle. [9][10][11][14][15][16][17][18][23][24][25] However, in these studies, the proteins collected from 8-10 volunteers are pooled in order to obtain enough amount of proteins to be analyzed by mass spectrometry, i.e., it is not possible   Classification of proteins according to: General Function: a) metabolism; b) biological process; c) transport; d) structure and structural organization; e) information pathways; f) miscellanea; Function in AP: g) metabolism; h) tissue regeneration; i) antimicrobial; j) immune response; k) lubrication; l) biomineralization; m) unknown biological function; Origin: n) cytoplasm origin; o) extracellular origin; p) nucleus origin; q) cytoskeleton origin; r) intracellular origin; s) membrane origin; t) unknown protein origin; Interaction: u) protein/protein interaction; v) calcium/phosphate binding; w) other molecular interaction; x) unknown molecular interaction. The groups are: 3% citric acid (CA), 0.5% sodium lauryl sulfate (SLS), 3% citric acid plus 0.5% sodium lauryl sulfate (CA+SLS) and 0.5% Sodium lauryl sulfate plus 3% citric acid (SLS+C).  to collection AEP proteins in vivo in order to perform immunoblotting analysis. 20 Since SDS is biocompatible and can be used to collected AEP proteins in vivo, in the present study we evaluated both 3% citric acid and  Continued from previous page Optimizing the formation of the acquired enamel pellicle in vitro for proteomic analysis * Proteins exclusively identified in each group. Proteins highlighted in bold are typical of the acquired enamel pellicle. The groups are: 3% citric acid (CA), 0,5% sodium dodecyl sulfate (SDS), 3% citric acid plus 0,5% sodium dodecyl sulfate (CA+SDS) and 0,5% Sodium dodecyl sulfate plus 3% citric acid (SDS+CA).