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LenzMass SpectrometrydiaPASEFBottom-up proteomicsDental erosionSalivary pellicleSmokerProteomeSalivahttps://www.ebi.ac.uk/pride/archive/projects/PXD055472SalivaSalivary composition Unstimulated and stimulated saliva samples were collected from all participants. Saliva samples were collected for 5 min at each of the three study visits (between 8 and 12 a.m.). For the collection of stimulated saliva, participants chewed a paraffin pellet (MD2425, AUROSAN). Saliva sample preparation for proteome analysis Aliquots containing 15 µg protein equivalent were reduced and alkylated with 10 mM tris(2-carboxyethyl)phosphine (TCEP) and 40 mM iodoacetamide (IAA) in a thermoshaker (30 min, 37°C, 600 rpm). Samples were digested with trypsin using a modified SP3 protocol in 96 well format on a robotic platform (KingFisher Duo Prime, Thermo Fisher Scientific). In brief, proteins were precipitated onto amine-coated paramagnetic beads (MagReSyn Amine, Resyn Biosciences) by adjusting to 70 % acetonitrile (ACN), washed 3 times with 100 % ACN, twice with 70 % ethanol, and digested with trypsin (Trypsin Gold, Promega; 1:10 substrate/enzyme ratio) in 25 mM aqueous ammonium biocarbonate buffer. Following digestion, peptide mixtures were acified with 10% aqueous formic acid (FA) and 100 % ACN to final concentrations of 0.1 % FA and 2 % ACN, respectively, spiked with a calibration peptide mixture (iRT Peptides, Biognosys; 1:100 dilution) and kept at 4°C for mass spectrometric analysis. Mass spectrometry-based proteome profiling by data-independent acquisition mass spectrometry (DIA MS) Proteome profiling was performed based on 250 saliva and 50 salivary pellicle samples. Two technical replicates per biological replicate were acquired. Samples were analyzed on a nanoflow chromatography system (nanoRSLC, Thermo Fisher Scientific) hyphenated to a hybrid timed ion mobility-quadrupole-time of flight mass spectrometer (timsTOF Pro 2, Bruker). In brief, 150 ng equivalents of peptides were enriched on a reversed-phase C18 trapping column (0.3 cm × 300 µm, Thermo Fisher Scientific) and separated on a reversed-phase C18 column with an integrated CaptiveSpray Emitter (PepSep MAX, 15 cm × 150 µm, Bruker) using a 13 min linear gradient of 5-35 % ACN / 0.1 % FA (v:v) at 850 nl min-1 and a column temperature of 50°C. Data-independent acquisition (DIA) analysis was performed in diaPASEF mode using an 8×2 variable size window acquisition method from m/z 400 to 1,200 to include the 2+/3+/4+ population in the m/z-ion mobility plane. The collision energy was ramped linearly as a function of the mobility from 59 eV at 1/K0=1.5 Vs cm-2 to 20 eV at 1/K0=0.7 Vs cm-2.PrideProtein identification was achieved using the Pulsar algorithm in Spectronaut Software (version 19.1, Biognosys) using default settings against 50 randomly selected DIA files. Data were searched against the UniProtKB Homo sapiens reference proteome (revision 08-2023) augmented with a set of 51 known common laboratory contaminants at default settings. The proteomes of 15 commonly observed bacterial species from the oral microbiome were included to account for bacterial contributions. For quantitation, up to the 6 most abundant fragment ion traces per peptide, and up to the 10 most abundant peptides per protein were integrated and summed up to provide protein area values. Mass and retention time calibration as well as the corresponding extraction tolerances were dynamically determined. Both identification and quantification results were trimmed to a false discovery rate (FDR) of 1 % using a forward-and-reverse decoy database strategy.ProteomicsChristof LenzCore Facility Proteomics, Department of Clinical Chemistry, University Medical Center Goettingen, GermanyPARTIALHomo Sapiens (human)christof.lenz@mpibpc.mpg.de10.1016/J.JDENT.2026.106517Max Planck Institute for Biophysical ChemistryGermanyfalseSaliva and salivary pellicle composition and proteomic profile in smokers vs. non-smokers and its effect on dental erosion (saliva data)Aim: This study aimed to analyse the salivary composition and proteomic profile of saliva and the salivary pellicle in smokers compared to non-smokers, and to examine potential differences of the erosion-protective capacity of the salivary pellicle. Materials and Methods: Twenty-five smokers and 25 non-smokers were included. Unstimulated and stimulated saliva samples were analysed regarding flow rate, pH, buffer capacity, calcium, phosphate, fluoride, and protein content. Salivary pellicles were formed intraorally on resin specimens (n=8). Saliva and salivary pellicle samples were used for data-independent acquisition mass spectrometry. Proteins were identified using the UniProtKB Homo sapiens reference proteome. For the saliva samples, proteomes of 15 commonly observed bacterial species from the oral microbiome were also included. In an in situ experiment, the participants used intraoral splints loaded with bovine enamel and dentin specimens for 120 min (each n=4). Pellicle-covered specimens were extraorally eroded (1mL HCl, pH 2.3, 60 s). Calcium release into the acid was determined photometrically and compared to pellicle-free specimens (control, each n=30). Differentially abundant proteins between smokers and non-smokers were identified using t tests (≥1.5-fold differences and padj.≤0.05). In addition, a pathway enrichment analysis of the associated genes was performed (KEGG, Reactome). Statistical analysis of the salivary samples was performed by one-way ANOVAs followed by pairwise t tests, t tests, Pearson correlations, and multiple linear regression analysis (p<0.05). Results: Except for phosphate in stimulated saliva (smokers: 5.5±2.1 mmol/L, non-smokers: 3.6±1.0 mmol/L; padj.=0.003), salivary parameters were not significantly different between smokers and non-smokers. For both saliva and the salivary pellicle, gene set enrichment analysis indicated common biological themes of genes enriched in either the smokers or the non-smokers. Despite the different proteomic profiles, the erosion-protective capacity of the salivary pellicle of smokers and non-smokers did not differ, and calcium release was not correlated with or predicted by the assessed salivary parameters. The salivary pellicle had a significant erosion-protective effect in enamel compared to the control (41.4±6.3 nmol/mm2), but no differences between smokers (33.2±10.6 nmol/mm2, padj.=0.001 ) and non-smokers (32.7±8.6 nmol/mm2, padj.=0.001) were found. Calcium release did not correlate with or was predicted by the assessed salivary parameters. Conclusion: The proteomic profiles of both unstimulated and stimulated saliva and the salivary pellicle differ between smokers and non-smokers. Evidence for different activity of different biological metabolic pathways such as carcinogesis exists. However, there was no detectable influence on the erosion-protective capacity of the pellicle.2026-06-302024-09-02PXD055472NEWT:330879NEWT:377960NEWT:1131NEWT:2042546NEWT:259447NEWT:295546NCBITaxon:2719036NEWT:1334565NEWT:625351NEWT:258594NCBITaxon:1280NEWT:112503NEWT:1129NEWT:309807NEWT:59753NEWT:89184NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:193516NEWT:1073882NEWT:6819NEWT:475174NEWT:111205NEWT:1117NEWT:498257NEWT:10036NEWT:1590NEWT:10034NEWT:33114NEWT:661410NEWT:638632NEWT:224326NEWT:376619NCBITaxon:79857NEWT:1096976NEWT:3765NEWT:3767NEWT:1589NEWT:135622NEWT:35786NEWT:1580NEWT:399784NEWT:96731NEWT:656064NEWT:1582NEWT:1584NEWT:36630NEWT:383379NEWT:418106NCBITaxon:12721NEWT:10029NEWT:913645NEWT:2492962NEWT:641809NEWT:1392488NEWT:38815NEWT:317447NEWT:4688NEWT:111225NEWT:7719NEWT:868565NEWT:135674NEWT:79329NEWT:30069NEWT:12637NEWT:59729NEWT:2164133NEWT:295105NEWT:108061NEWT:60711NEWT:224308NEWT:3347NEWT:160621NEWT:212790NEWT:110368NEWT:55350NEWT:1310161NEWT:77133NEWT:145481NEWT:1310165NEWT:29058NCBITaxon:79824NEWT:1912919NEWT:410658NEWT:5516NEWT:44688NEWT:44689NEWT:498211NEWT:347256NEWT:5518NEWT:398007NEWT:1527468NEWT:498217NEWT:498216NEWT:11320NEWT:246196NEWT:246197NEWT:145458NEWT:1310173NEWT:44685NEWT:161934NEWT:156587NEWT:1148NEWT:5508NEWT:3329NEWT:419481NEWT:5507NEWT:410661NEWT:1140NCBITaxon:2157NEWT:1143NEWT:1287689NEWT:1094343NEWT:1462472NEWT:1336795NEWT:644042NEWT:150808NEWT:1182590NEWT:3712NEWT:3711NEWT:3714NCBITaxon:37162NEWT:270643NEWT:2065263NEWT:177437NEWT:10418NEWT:118698NEWT:1616117NEWT:118696NEWT:34865NEWT:52283NEWT:28174NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:44664NEWT:43346NEWT:3702NEWT:1245466NEWT:3704NEWT:244366NEWT:1246791NEWT:176597NEWT:118694NEWT:2850NEWT:118691NEWT:34871NEWT:33548NEWT:52299NEWT:8165NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:926571NEWT:536231NEWT:376219NEWT:219813NEWT:1510NEWT:460519NEWT:1515NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:1348799NEWT:33995NEWT:55784NEWT:1303443NEWT:9483NEWT:485NEWT:56636NEWT:2709072NEWT:2499525NEWT:2853422NEWT:487NEWT:1538553NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:1185650NEWT:216778NEWT:294607NEWT:1502NEWT:128017NEWT:376686NEWT:237610NEWT:95486NEWT:1883446NEWT:1233435NEWT:118223NEWT:11757NEWT:987059NEWT:109760NEWT:29031NEWT:317047NEWT:491NEWT:460523NEWT:45954NEWT:235443NEWT:1757312NEWT:85694NEWT:109786NEWT:108458NEWT:5936NEWT:320637NEWT:1078283NEWT:3750NEWT:983964NEWT:11706NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:3747NEWT:1116234NEWT:1255228NEWT:410289NEWT:373153NEWT:1640278NEWT:472NEWT:940825NEWT:529507NEWT:1071661NEWT:28128NEWT:470NEWT:5911NEWT:748693NCBITaxon:50557NEWT:39251NEWT:29491NEWT:101841NEWT:446NEWT:153481NEWT:2014887NEWT:33952NEWT:445NEWT:153009NEWT:261756NEWT:166313NEWT:63366NEWT:63367NEWT:215402NEWT:1547NEWT:1290454NEWT:906914NEWT:28141NEWT:9031NEWT:27292NEWT:1050722NEWT:3728NEWT:108931NEWT:1293497NEWT:1055524NEWT:150475NEWT:267872NEWT:381666NEWT:172269NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:8694NEWT:33936NEWT:8692NEWT:2903NEWT:185579NEWT:13076NEWT:207340NEWT:1006581NEWT:8673NEWT:33940NEWT:550NEWT:554NEWT:451516NEWT:552NEWT:1325291NEWT:42410NEWT:36185NEWT:1054211NEWT:1225786NEWT:771870NEWT:51338NEWT:575412NEWT:28112NEWT:49012NEWT:6493NEWT:10832NEWT:6494NEWT:6491NEWT:507601NEWT:520NEWT:186441NEWT:643680NEWT:214092NCBITaxon:6157NEWT:13095NEWT:162425NEWT:104105NEWT:216257NEWT:9986NEWT:8654NEWT:8658NEWT:1268063NEWT:8657NEWT:9983NEWT:8655NEWT:5147NEWT:595495NEWT:28108NEWT:28104NEWT:407821NEWT:568703NCBITaxon:2NEWT:568708NEWT:1613NEWT:986NEWT:52641NEWT:28532NEWT:353152NEWT:298176NEWT:353153NEWT:227377NEWT:1774284NEWT:40674NEWT:1194669NEWT:51329NEWT:443906NEWT:13037NEWT:519NEWT:2510939NEWT:7394NEWT:6063NEWT:1328388NEWT:105487NEWT:1548728NEWT:667127NEWT:9557NEWT:377586NEWT:300641NEWT:39655NEWT:9554NEWT:38323NEWT:475932NEWT:256318NEWT:206411NCBITaxon:6191NEWT:229533NEWT:214053NEWT:28550NEWT:80863NEWT:90675NEWT:52639NEWT:52638NEWT:57075NEWT:1666905NEWT:8697NEWT:861109NEWT:8695NEWT:884204NEWT:510516NEWT:1045775NEWT:1123869NEWT:9544NEWT:9545NEWT:979NEWT:7370NEWT:83906NEWT:99488NEWT:1134506NEWT:255470NEWT:38783NEWT:6426NEWT:33090NEWT:9935NEWT:287889NEWT:305959NEWT:1028307NEWT:13489NEWT:29829NEWT:92867NEWT:92866NCBITaxon:3055NEWT:51750NEWT:202950NEWT:441772NEWT:1293091NEWT:48106NEWT:202956NEWT:295027NCBITaxon:11320NEWT:632957NEWT:9925NCBITaxon:9606NEWT:90690NEWT:1436183NEWT:300273NEWT:4232NEWT:416348NEWT:11298NEWT:4236NEWT:7742NEWT:196627NEWT:1076878NEWT:200308NEWT:200302NEWT:870435NEWT:58370NEWT:9913NEWT:9915NEWT:105841NEWT:2666255NEWT:999810NCBITaxon:5693NEWT:28995NEWT:2144189NEWT:1392998NEWT:380394NEWT:1150621NEWT:226900NEWT:1266738NEWT:231490NEWT:244704NEWT:7725NEWT:9909NEWT:9902NEWT:430615NEWT:563041NEWT:72664NEWT:36599NEWT:41949NEWT:249161NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1678078NEWT:749906NEWT:418985NEWT:749907NEWT:150847NEWT:29835NEWT:431947NEWT:69014NEWT:142809NEWT:1095183NEWT:302409NEWT:130821NEWT:27606NEWT:1519788NEWT:59202NEWT:9975NEWT:8643NEWT:1801631NEWT:1159899NEWT:502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