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Original Article Proteomic Analysis of Whole Saliva in Relation to Dental Caries Resistance

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Original Article Proteomic Analysis of Whole Saliva in Relation to Dental Caries Resistance Original Article Proteomic Analysis of Whole Saliva in Relation to Dental Caries Resistance (dental caries / fractions of whole human saliva / label-free quantification / mass spectrometry / proteins) L. KulhavÁ1, 2, A. ECKHARDT2, S. Pataridis2, R. Foltán3, I. MIKŠÍK2 1Department of Analytical Chemistry, Faculty of Science, Charles University, Prague, Czech Republic 2Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic 3Department of Stomatology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic Abstract. Saliva contains possible biomarkers that Received: November 29, 2019. Accepted: March 10, 2020. are associated with dental caries. The present study This work was supported by Charles University, project GA UK aimed to analyse differences in the abundance of No. 322216, within the framework of Specific University Re- proteins in the saliva between caries-positive (CP; search (SVV 260560), and by the Czech Science Foundation (No. N = 15) and caries-free (CF; N = 12) males and to 17-31564A). compare differences in the abundance of proteins be- Corresponding author: Lucie Kulhavá, Institute of Physiology of tween two saliva sample fractions (supernatant and the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague pellet). We found 14 differently significantly ex- 4, Czech Republic. Phone: (+420) 241 06 2127; e-mail: lucie. pressed proteins in the CF group when comparing [email protected] the supernatant fractions of the CP and CF groups, Abbreviations: AMY1C – α-amylase 1 (IPI00300786), ALB – se- and three proteins in the pellet fractions had signifi- rum albumin (IPI00745872), AZGP1 – zinc-α-2-glycoprotein cantly higher expression in the CP group. Our results (IPI00166729), BPIFB1 – BPI fold-containing family B member indicate very specific protein compositions of the sa- 1 (IPI00291410), BPIFB2 – BPI fold-containing family B mem- liva in relation to dental caries resistance (the saliva ber 2 (IPI00296654), CA6 – carbonic anhydrase 6 (IPI00295105), of the CP group mainly contained pellet proteins and CSTB – cystatin-B (IPI00021828), CST1 – cystatin-SN (IPI00305477), CST4 – cystatin-S (IPI00032294), CRNN – cor- the saliva of the CF group mainly contained superna- nulin (IPI00297056), C6orf58 – protein LEG1 homologue tant proteins). This was the first time that the saliva (IPI00374315), FDR – false discovery rate, GO – gene ontology, pellet fraction was analysed in relation to the dental HSPB1 – heat-shock protein β 1 (IPI00025512), H3F3A – histone caries status. We detected specific calcium-binding H3.3 (IPI00909530), IGHA1 – immunoglobulin heavy constant α proteins that could have decalcified enamel in the sa- 1 (IPI00386879), Ig heavy chain V-III – immunglobulin heavy liva pellet of the CP group. We also observed signifi- chain V-III region (IPI00854644), IGJ – immunoglobulin J chain cantly up-regulated immune proteins in the saliva (IPI00947235), IGK@ IGK@ – IGK@ IGK@ protein supernatant of the CF group that could play an im- (IPI00784985), IGLV1-44 – immunoglobulin λ variable 1-44 portant role in the caries prevention. The particular (IPI00887169), JUP – junction plakoglobin (IPI00554711), KR- T6A – keratin, type II cytoskeletal 6A (IPI00909059), KRT1 – protein compositions of the saliva pellet and super- keratin, type II cytoskeletal 1 (IPI00220327), KRT4 – keratin, natant in the groups with different susceptibilities to type II cytoskeletal 4 (IPI01022175), KRT5 – keratin, type II cy- tooth decay is a promising finding for future research. toskeletal 5 (IPI00009867), KRT13 – keratin, type I cytoskeletal 13 (IPI00009866), KRT13 GUCA1B – KRT13 GUCA1B protein (IPI00930614), LCN1 – lipocalin-1 (IPI00009650), LTF – lac- Introduction totransferrin (IPI00925547), LYZ – lysozyme C (IPI00019038), Oral fluids are very important in oral health. The MS – mass spectrometry, PIGR – polymeric immunoglobulin re- physiology of human saliva and salivary secretion were ceptor (IPI00004573), PIP – prolactin-inducible protein reviewed in Proctor (2016). Saliva includes many mark- (IPI00022974), RAB10 – Ras-related protein Rab 10 (IPI00016513), S100A8 – protein S100-A8 (IPI00007047), ers that can indicate a risk of certain diseases (Podzimek S100A9 – protein S100-A9 (IPI00027462), S100A14 – protein et al., 2016). Possible biomarkers for dental caries, such S100-A14 (IPI00010214), TF – serotransferrin (IPI00022463), as salivary electrolytes, microorganisms, proteins and TGM3 – protein-glutamine γ-glutamyltransferase E (IPI00300376), peptides, were also previously reviewed along with the ZG16B – zymogen granule protein (IPI00060800). functional properties of saliva (Gao et al., 2016). Only a Folia Biologica (Praha) 66, 72-80 (2020) Vol. 66 Pellet in Proteomic Analysis of Saliva 73 small part of the population at the age of 30 is classified tease activity. The unstimulated whole-saliva samples as caries-free, and the reason for the resistance is un- were frozen at –80 °C until further analysis. known. There are limited studies on the differences in the saliva protein composition between persons with Initial sample preparation carious teeth and persons without caries (Al-Tarawneh The saliva samples (700 μl) were centrifuged at et al., 2011; Laputková et al., 2018). Differences in the 13,000 g for 30 min at 4 °C. The supernatant and pellet abundance of salivary proteins between caries-suscepti- (P) of each sample were retained separately. The ob- ble and caries-free persons and a gender comparison tained supernatants and pellets were then aliquoted for were presented in our previous study (Kulhavá et al., use. Each sample was divided into seven parts; four 2018). Age-specific variations in the salivary proteome parts were used for two-dimensional electrophoresis, of caries-susceptible adults and elderly people have also two parts were used for two-dimensional difference gel been investigated (Wang et al., 2018a). The influence of electrophoresis and one part was used for MS analysis salivary protein composition on in vitro dental pellicle and label-free (LF) quantification. The pellets were ly- and their correlation with dental caries was studied by ophilised before distribution. Seven samples (super- two-dimensional electrophoresis (Vitorino et al., 2006). natant/pellet) of human saliva (caries-positive, N = 7; Salivary protein roles as predictors of caries risk were caries-free, N = 7) were used for two-dimensional elec- described in a recent review (Laputková et al., 2018). trophoresis because high variability between each saliva However, studies on the protein composition of saliva sample was identified (verified by the previous one-di- in relation to dental caries have varied (different ap- mensional electrophoresis), along with different protein proaches, methods, and use of gender-based compari- concentrations in individual saliva samples. Four sam- sons). A study by Vitorino et al. described an assessment ples (supernatant/pellet) of human saliva (caries-posi- of pre-treatment saliva samples using in-gel and off-gel tive, N = 4; caries-free, N = 4) were analysed by two- approaches, and they were the first to present the pellet dimensional difference gel electrophoresis because of fraction protein content (but not related to caries) the cost of access method. (Vitorino et al., 2012). All comparative studies of caries- positive and caries-free individuals have analysed only Preparation of samples for two-dimensional the supernatant saliva fraction. The present study, based electrophoresis and two-dimensional difference on analyses by mass spectrometry (MS) (and label-free gel electrophoresis mass spectrometry quantification), was performed to compare differences in the salivary protein abundance Supernatant proteins were precipitated using a final between caries-free and caries-positive males. We com- concentration of 10% (v/v) trichloroacetic acid and pared differences in the abundance of proteins in two 0.12% (w/v) dithiothreitol (Jehmlich et al., 2013). After saliva sample fractions: the supernatant and the pellet. vortexing followed by incubation at 25 °C for 15 min, Here, the pellets were analysed in this way for the first the precipitated protein was concentrated by centrifuga- time, and a unique comparison between these saliva tion (13,000 g, 15 min, 4 °C). Then we used a protocol fractions was carried out. modified from Gonçalves et al. (2011); ice-cold acetone was used without dithiothreitol. Protein pellets from the Material and Methods collected supernatants (S) were washed three times with ice-cold 100% acetone, lyophilised and stored at –80 °C. Saliva sample collection Pellet proteins (P) were washed with ice-cold acetone and acetonitrile, lyophilised and stored –80 °C. Unstimulated whole saliva was collected from 27 healthy male non-smoking volunteers (without diabetes MS sample preparation – supernatant (S) and mellitus). The subjects were categorized according to pellet (P) protein the DMFT index (decayed, missing, and filled teeth in- dex) into two groups: caries-positive (CP) (N = 15, aged Protein from supernatant and pellet samples was ob- 38.4 ± 5.6, DMFT ranging from 7 to 12) and caries-free tained similarly as in the sample preparation for the two- (CF) (N = 12, aged 31.8 ± 7.6, DMFT ranging from 0 (N dimensional gel electrophoresis. Pellet proteins were = 10) to 1(N= 2)). All procedures performed in the stud- digested in a solution containing 0.1 M NH4HCO3 and ies involving human participants were in accordance 0.2 M trypsin
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