Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/PXD011318_SupplementaryTable2_phosphopeptideList.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/PXD011318_SupplementaryTable1_proteinList.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_Phospho_Re-Seach_090618.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_3_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_5_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_3_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_2_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_6_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_2_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_6_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_4_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_5_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_4_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_1_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_2_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_6_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_4_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_1_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_5_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_3_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_3_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_5_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_6_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_1_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_4_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_2_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_4_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_6_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Phospho_1_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_3_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_5_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_3_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_5_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_4_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_2_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_6_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_2_R1_180824033256.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_1_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP_1_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_Phospho_Re-Search_090618.pdResultftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/07/PXD011318/OKF6_Shisha_TP.pdResultprimaryOK200aditixchatterjee@gmail.comAditi ChatterjeeMass SpectrometryShotgun proteomicsNot availableHigh-throughputOsccOral cancerHookahWaterpipe tobacco smokingCarcinogenesisNarghile tobaccoOrbitrap fusionhttp://www.ebi.ac.uk/pride/archive/projects/PXD011318Oral EpitheliumOKF6/TERT1 were grown and maintained in keratinocyte serum free medium (KSFM) (supplemented with bovine pituitary extract (25 µg/ml), epidermal growth factor (EGF) (0.2 ng/ml) (ThermoFisher Scientific, MA), 1% penicillin/streptomycin and calcium chloride (0.4 mM). The cells were cultured at 37°C in a humidified air incubator with 5% CO2. Shisha extract was prepared and used to treat OKF6/TERT1 cells chronically for up to a period of 8 months. OKF6/TERT1-Parental and OKF6/TERT1-Shisha-8M cells were grown to 80% confluence followed by serum starvation for 8 h. The cells were washed with 1X PBS thrice and harvested in lysis buffer (2% SDS, 5 mM sodium fluoride, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate in 50 mM Triethyl ammonium bicarbonate (TEABC)). The cell lysates were sonicated, centrifuged and protein concentration was determined by BCA (Thermo Scientific, Bremen, Germany). Equal amount of protein was collected from both conditions for further processing. Samples were reduced using dithiothreitol (DTT) at 60ºC for 20 min and alkylated using iodoacetamide (IAA) for 10 min at room temperature. The samples were then precipitated using 6 volumes of chilled acetone at -80oC overnight. Following this, the samples were centrifuged for 15 min at 12,000 rpm, acetone was removed and the pellet was air-dried. This was followed by reconstitution of protein pellets from both conditions in 4M urea. Protein samples were digested using Lysyl Endopeptidase®, Mass Spectrometry Grade (Catalog#125-05061, Wako, Richmond, VA) in an enzyme-to-protein ratio of 1:100 for 4 h at 37 o C. Subsequently, the urea concentration was reduced from 4M to 2M using 50mM TEABC. The samples were then digested using TPCK-treated trypsin (Worthington, NJ) at an enzyme-to-protein ratio of 1:20 for 16 h at 37ºC. This was followed by sample clean-up using Sep-Pak Classic C18 columns (Catalog#WAT051910, Waters, Milford, MA). The samples were then vacuum-dried and reconstituted in 50mM TEABC for TMT-labeling. Peptide samples were labelled using Tandem Mass Tag (TMT) labels (ThermoScientific, Bremen, Germany) as per manufacturer’s instructions. Peptides derived from OKF6/TERT1- Parental and OKF6/TERT1-Shisha-8M were labelled with TMT tags 130C and 131, respectively. TMT labelled peptides were pooled and subjected to basic pH reverse phase chromatography (bRPLC), as previously described. The 96 fractions obtained were concatenated into 6 fractions. From the pooled fractions, one-tenth volume equivalent peptides were taken for total proteome analysis. The remaining samples were subjected to immobilized metal affinity chromatography (IMAC) based phosphopeptide enrichment. The enriched samples were desalted and used for mass spectrometry-based analysis. Both phosphopeptide enriched and total proteome fractions were analysed on Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Bremen, Germany) interfaced with Easy nLC-1000 system (Thermo Scientific, Odense, Denmark). The peptide fractions were reconstituted in 0.1% formic acid (Solvent A) and loaded onto a trap column (75 μm x 2 cm, Magic C18AQ, 5 μm, 100 Å, Michrom Biosciences Inc., Auburn, CA) at a flow rate of 3 µl/min. The peptides were then resolved on an in-house packed analytical column 75 μm x 20 cm, Magic C18 AQ, 3 μm, 100 Å, (Michrom Biosciences Inc., Auburn, CA) at a flow rate of 300 nl/min with back pressure of 275 bar using a gradient of 10% –35% solvent B (0.1% formic acid in 95% acetonitrile) for 100 min. Data acquisition on the Orbitrap Fusion was carried out using a data-dependent method with MS 2 scanning for TMT tags. The scan sequence was started with the acquisition of a full MS 1 spectrum acquired in the Orbitrap (m/z range, 350-1600; resolution, 1.2x10 5 ; AGC target, 4x10 5 ; maximum injection time, 50 ms). MS2 scans were done in Orbitrap mass analyser using the following settings: quadrupole isolation at an isolation width of 1.6 m/z; fragmentation method, HCD; normalized collision energy, 34%). The peptide fractions were analysed in triplicate on the mass spectrometer.PrideTMTphosphorylated residueiodoacetamide derivatized residueTMT6plex-126 reporter+balance reagent acylated residuemonohydroxylated residueIdentification and quantification of proteins was carried out using Proteome Discoverer (version 2.1) software suite (Thermo Scientific, Bremen, Germany). Raw files were searched against NCBI human RefSeq protein database (version 81) containing common contaminants, using Sequest HT search algorithm. In the search parameters, carbamidomethylation of cysteine, and TMT label at peptide N-termini and lysine were set as static modifications; while oxidation of methionine was set as a dynamic modification. For phosphoproteomic data, phosphorylation of serine, threonine, and tyrosine were defined as additional dynamic modifications. Trypsin was chosen as the protease with a maximum allowed missed cleavage of 2. Precursor mass tolerance was set to 10 ppm and fragment mass tolerance was set to 0.05 Da. Decoy database search was carried out and peptide spectrum matches with a false discovery rate (FDR) cut-off of 1% was considered for peptide identification.ProteomicsAditi ChatterjeeOrbitrap FusionPARTIALInstitute of Bioinformatics, 7th floor, Discoverer building, International Tech Park, Bangalore 560 066Homo Sapiens (human)31321732 Patil S, Rajagopalan P, Patel K, Subbannayya T, Babu N, Mohan SV, Advani J, Sathe G, Bhandi S, Solanki HS, Sidransky D, Chatterjee A, Gowda H, Ferrari M. Chronic shisha exposure alters phosphoproteome of oral keratinocytes. J Cell Commun Signal. 2019 10.1007/s12079-019-00528-4aditixchatterjee@gmail.comBiologicalBiomedicalInstitute of Bioinformatics, Bangalore, Karnataka, IndiaIndiaShisha smoking has been epidemiologically linked to oral cancer. However, few studies have investigated the pathobiology of shisha-induced cellular transformation. We studied the effects of chronic shisha exposure (8 months) in an in vitro model using immortalized, non-neoplastic oral keratinocytes (OKF6/TERT1). Quantitative proteomic and phosphoproteomic analyses were performed on OKF6/TERT1 cells treated with shisha extract for a period of 8 months. Pathway analysis was carried out to identify significantly enriched biological processes in shisha-treated cells. Chronic shisha exposure resulted in increased cell scattering phenomenon in OKF6/TERT1 cells. Data analysis revealed differential phosphorylation of 164 peptides (fold change ≥1.5, p ≤ 0.0.5) corresponding to 136 proteins. Proteins associated with mTORC1 and EIF4F complexes involved in initiating protein translation were seen to be enriched upon shisha treatment. Network analysis also highlighted downregulation of proteins involved in Type I interferon signaling in shisha-treated cells. Quantitative phosphoproteomic approach elucidated global perturbations to the molecular milieu of oral keratinocytes upon shisha exposure. Further studies are needed to validate putative targets in oral cancer patients with shisha smoking history.Chronic shisha exposure alters phosphoproteome of oral keratinocytes.Patil Shankargouda S, Rajagopalan Pavithra P, Patel Krishna K, Subbannayya Tejaswini T, Babu Niraj N, Mohan Sonali V SV, Advani Jayshree J, Sathe Gajanan G, Bhandi Shilpa S, Solanki Hitendra S HS, Sidransky David D, Chatterjee Aditi A, Gowda Harsha H, Ferrari Marco Msodium salt, dairy cow, hypophysis cerebri, DmelCG8566, ammonium formate, bar-h1, 13C-labeled, cadmium salt, MeCN, PLXN5, Elp-B1, der, Elp-1, bar, SDH, 2-(indol-3-yl)ethanoic acid, Basodexan, magnesium formate, zinc salt, Solvent, Long Term, SeP, pituitary, Bca, dmTAF[[II]]230, 2-Propanone, SDS, Polypeptides, protein polypeptide chains, 1H-imidazoleacetic acid, CEH, cobalt(II) formate dihydrate, CH3-C#N, B1, 1, Urea, HRR-1, 2, NEPII, AGM4, Barh1, Analysis, Sodium Ion, SEH, Faroe Islands, Fluoristat, WMS, Sep, SEP, O-2-deoxy-2-(methylamino)-alpha-L-glucopyranosyl-(1-2)-O-5-deoxy-3-C-formyl-alpha-L-lyxofuranosyl-(1-4)-N, (R*, bca, sarcoma of breast, TFIID TAF250, Analyses, N'-bis(aminoiminomethyl)-, cel, Fluoride, proteins, flb, sEP, AI047805, isolation and purification, Bos Tauurus, Strepto-Hefa, IMAC, oxen, decreased, column, Magics, sample, D1, keratinized cell of epidermis, autolysin activity, imac, RIP14, SGS, nickel salt, B2AR, dTAF[[II]]230, Elp, HRR1, BZRP, cobalt (+2) salt, DEgfr, TAF200, Longterm Effect, Penicillin, breast sarcoma, Spectrum Analysis, not genetically inherited, CG4637, PBS, ACMICD, PBR, torpedo/egfr, (Indol-3-yl)acetate, sodium (4:1:1) salt, Dm1, magnesium salt, BH1, sodium, Streptomycine Panpharma, hrr1, Urogastrone, natrium, Estreptomicina Normon, egfr, HD-33, El, LY57, DER/EGFR, EGFR, EGfr, EgfR, formate, Spectrometry, DER flb, pooled, cromium (+3), Torpedo/Egfr, Ossin, beta Trypsin, Degfr, Indole-3-acetic acid, Painful bladder syndrome, heteroauxin, SCYA26, Taf250, DEGFR, l(2)05351, bacteriolytic toxin activity, ammonium (4:1) salt, methyl cyanide, bovine, lead (+2) salt, TMT, TAF230, hg, BPBS, HH, Cleland Reagent, F10B6_15, EGF receptor binding, Dunc104, D-Egf, nickel formate dihydrate, proto-oncogene c-ErbB-1, lead formate, sarcoma of the breast, Effects, Streptomycin Grünenthal, aluminum salt, 4-dimercapto-, Strepto Fatol, Carbamide, R*)-, EK2-6, protein-containing complex, thomson, body system, CG5529, F10B6.15, CG7826, Hh, mass-to-charge ratio, Ion Level, period, beta-Urogastrone, RNF47, Hp, isolation, AI790464, Ly57, EGFr, CG7835, CG42273, torpedo/Egfr, system, TOP, Growth Factor, Carmol, Catalogs, transforming growth factor alpha receptor ligand, anatomical systems, dTAF[[II]]250, epidermal growth factor receptor ligand, URG, thallium (+1) salt, Longterm, cell, beta-Trypsin, rubidium salt, top, 4733401P19Rik, pk18, Long-Term, methanoic acid, MBR, CD156, Injectable, dTAF250, Fluoristats, Faeroe Islands, xfor, bar-3, PTBR, peptidos, Antibiotics, Chromatographies, Anhydrous, PTHB1, Elp-B1RB1, 1H-indol-3-ylacetic acid, 1-(14)C-labeled, cow, dEGFR1, ERBB, barh1, Clelands Reagent, Mell1, BG:DS00004.13, potassium formate, copper (+2) salt, acetonitrile, buffer, Cell, dTAF230, polypeptide, hrr-1, transforming growth factor alpha, native protein, Temperatures, DmelCG5529, carbonyldiamide, Bos primigenius taurus, C18, cupric formate, chemical analysis, barH1, Long Term Effects, TAF[[II]]250/230, MFS1, domestic cattle, Sodium, DER/top, aluminum formate, Mass Spectrum Analyses, Sodium Ion Level, Cleland's, Achromobacter protease I, Taf[[II]]250, AU020952, FXR, lithium formate, holin, underdeveloped, 3-Indolylessigsaeure, Na, SWDS, Natrium, MIP-4alpha, Klp53D, ethanenitrile, Keratinocyte, mDRC, CD156a, primary structure of sequence macromolecule, Longterm Effects, ME-IV, achromopeptidase, plan specification, Cleland's Reagent, fxr, Epidermal, ox, anon-WO0182946.19, Sodium 23, ADRB2R, NEP2, DER/faint little ball, NCMe, determination, Blood, pituitary body, Nl1, zinc formate, TGF-alpha receptor binding, lead salt, protein, DmKlp53D, Serum, lysis, extracted material, Bar H1, peptide, BETA2AR, Incubator, sodio, ammonium tetraformate, Streptomycin Sulfate, anon-WO0134654.19, IAA, ClvPrd, peptido, DmelCG4637, Human Urinary Gastric Inhibitor, NL1, Min, NL2, Mir, d-egf-r, protein aggregate, present in fewer numbers in organism, Injectables, Effect, DmelCG42273, Penicillin Antibiotics, Mass Spectrum Analysis, dermoid cyst with malignant transformation, peptides, E927b, Bos bovis, E430039A18Rik, hypoplasia, min, mAPC, achromobacter beta-lytic protease (blp), Growth Factor-Urogastrone, IBP, purification, copper, SCAN, free, glandula pituitaria, transforming growth factor alpha receptor binding, lithium salt, SOLO DANCERS, Sputolysin, D-EGFR, domestic cow, necrosis, Lyw-57, Bladder pain syndrome, GPHYSD2, ammonium (2:1) salt, Long-Term Effects, DER1, l(2)09261, rip14, TSC-1, Dmel_CG7826, AI836084, Th, Blood Serum, TAFII-250, TAF250/230, KIF1B, Thermo Scientific, CG10079, ur, Spectroscopy, SLP65, Sodium-23, Ly-57, carbamide, TAFII250, S-adenosyl-L-methionine:thiol S-methyltransferase activity, HCD, Cleland, Streptomycin Sulphate, MMEL2, Fluorides, ADRBR, beta Urogastrone, bar3, lysyl endoprotease, bacteriocin activity, IES, CG8566, nickel (+2) salt, Dmel_CG7835, Mnb, MNB, imidazole-4-acetic acid hydrochloride, SLP-65, ammonium salt, Painful Bladder Syndrome, l(3)neo56, l(3)neo57, BASH, MS/MS, dEgfr, cobaltous formate, Epidermal growth factor, 3-Butanediol, MASS, AW124434, CG17603, TAF[[II]], organ system, Calcium Chloride, H2NC(O)NH2, DmCG8566, lysin activity, SR3-5, Acetamide, l(2)57DEFa, copper salt, Calcium chloride, dEGFR, Eph2, Polypeptide, DmHD-33, TGFalpha receptor binding, time, 1728, Proteomes, Bladder Pain Syndrome, receptor tyrosine-protein kinase erbB-1, malpighian cell, d230, C-erb, Reagent, cesium salt, mor1, FBN, dTAFII250, PLEXIN-B1, Spectrum Analyses, hypophysis, EfW1, Buffer, formic acid, Karbamid, method, potassium salt, Bar-H1, Sodium Fluorides, polypeptide chain, reduced, cattle, Strepto-Fatol, dmTAF1, Taf230, subnumerary, ECTOL1, Injection, 14C-labeled, method used in an experiment, c-erbB, Harnstoff, Mass, Strepto Hefa, Egf-r, tiny, Level, epidermal growth factor, Mass Spectroscopy, TAF250, EGF-R, Taf200, CG2916, D-Streptamine, MS2, Mrt, EGFR binding, imidazolyl-4-acetic acid, Torpedo/DER, Taf1p, Indoleacetic acid, labeling, decreased number, calcium formate, top/flb, OCTD, Epidermal Growth Factor-Urogastrone, Estreptomicina CEPA, HOMG4, (indol-3-yl)acetic acid, MIP-4a, Zymafluors, l(2)57EFa, EGF receptor ligand, SELP, l(2)57Ea, cromium (+3) salt, Long-Term Effect, TAF, 2-iodo-, sodium formate, thiol methyltransferase activity, BLNK-S, small, DYRK1, l(3)hh, data, TAF[[II]]250, ng/ml, nickel formate, DBI, 3H-labeled, protein complex, imidazole-4-acetic acid, total expressed protein, strontium formate, l(3)84Ab, strontium salt, Peptide, sep5, DmelCG10079, PKBS, natural protein, p230, Unc104, Protein, sequence, TFIID, Dyrk1, Vacuums, connected anatomical system, ACETONITRILE, tandem MS, WMS2, cyanomethane, teratoma with malignant transformation, Mass Spectrum, Ossins, TAF[[II]]230, top/DER, Calcium Chloride Dihydrate, Streptomycin Sulfate (2:3) Salt, uree, pituitary gland, urea, CC1, DmelCG2916, Tripcellim, ERBB1, TAF[II]250, CGI-97, sample population, Unc-104, chromic formate, UREA, Trypure, 11Na, EGF, Egf, Zymafluor, EFG-R, DmelCG17603, Pressures, concentration, Estreptomicina Clariana, Sodium fluoride (NaF), SSKS, Der, DER, calcium salt, Bar, Peptid, BAR, assay, BarHI, Peptide., Serums, DER/torpedo, HER1, TAF1high time, Chronic, slow time, prolonged period, increased period, increased time, chronic, Keratinocyte.Bru, IPP2A2, para Tyrosine, Raw, Lysine Hydrochloride, cleavage, L-Threonine, protein, phosphorylation, 2-Amino-3-hydroxypropionic acid, 5730420M11Rik, peptide, Polypeptides, Cut, protein polypeptide chains, L-Isomer Methionine, Lysine Acetate, Methionine, Drug Tolerance, ClvPrd, peptido, l(1)7Ba, l(1)7Bb, 2, Software Engineering, protein aggregate, Computer Program, WMS, Threonin, L Lysine, Svc, SET, F, dermoid cyst with malignant transformation, peptides, K, TAF-I, M, Open, phosphatase 2A inhibitor I2PP2A, 2-amino-3-(4-hydroxyphenyl)propanoic acid, 3-(p-Hydroxyphenyl)alanine, Computer Programs and Programming, epsilon-diaminocaproic acid, proteins, Y, CG11387, DmelCG4299, set, IGAAD, LYS, 2-amino-3-hydroxypropanoic acid, DmelCG10574, Lysin, Tyr, ppm, CT, Half Cystine, GPHYSD2, Racemethionine, drug tolerance, SGS, ratio, phapii, Zinc Cysteinate, immune system tolerance, T5E21.12, 2-Amino-4-(methylthio)butyric acid, 2-Amino-3-(p-hydroxyphenyl)propionic acid, Ct, StF-IT-1, para-Tyrosine, Thermo Scientific, Source Softwares, Software Tools, Programs, Tyrosine, ACMICD, Program, Computer Applications, S-adenosyl-L-methionine:thiol S-methyltransferase activity, 3-Hydroxyalanine, Algorithm, Computer Applications Software, Computer Applications Softwares, lysine, Immunologic Tolerance, Methionin, methionine, Softwares, Hmet, BcDNA:GH10590, tyrosine, T5E21_12, parent ion, Software Applications, Serine, HLA-DR-associated protein II, DI-2, Source Software, I-2Dm, L-isomer, proportionality, common, rate, L-Cysteine, alpha, CG4299, MASS, inhibitor of granzyme A-activated DNase, beta Trypsin, human, L isomer, I-2PP1, Applications, TAF-IBETA, Self Tolerance, precursor ion, Tolerance, L Serine, TAF-Ibeta, L-Tyrosine, Polypeptide, L-Serine, TMT, i2pp2a, Liquimeth, Computer Software Applications, human being, Cysteine Hydrochloride, 2-amino-4-(methylsulfanyl)butanoic acid, l(1)VE614, False, L Isomer, FBN, Gene, Computer, precursor, protein-containing complex, PHAPII, polypeptide chain, template-activating factor I, ECTOL1, Gene Products, L-Methionine, Del(8)44H, Lysine, Half-Cystine, Pedameth, Application, Open Source Softwares, proportion, Software Application, L Cysteine, ipp2a2, beta-Trypsin, Open Source Software, 2pp2a, L Threonine, man, kf, CG10574, Computer Software Application, OCTD, 2PP2A, 2-amino-4-(methylthio)butanoic acid, Tools, 10^[-6], DmelCG11387, L Tyrosine, taf-ibeta, dSET, dSet, peptidos, Tyrosin, metionina, thiol methyltransferase activity, Applications Software, serine, Open Source, data, Computer Software, DL-Methionine, protein complex, Proteins, igaad, Phosphorylations, L-Isomer, Peptide, Tool, polypeptide, Serin, Software Tool, native protein, Immune Tolerance, natural protein, I-2PP2A, tirosina, Acetate, Protein, Dm I-2, I2PP2A, MFS1, Software, L-Lysine, Data Base, Met, WMS2, teratoma with malignant transformation, Col4a-1, Engineering, Tripcellim, Protein Gene Products, incised, Computer Programs, Gene Proteins, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, Applications Softwares, SSKS, alpha-amino-gamma-methylmercaptobutyric acid, quotient, Peptid, threonine, 6-diaminohexanoic acid, AA407739, Enisyl, Peptide., Immunological Toleranceprojections, nPKC-delta, ORAL NEOPL, malignant Growth, protein translation, Cancer of the Mouth, neoplasm of tongue, Gene Ontology Projects, single-organism developmental process, determination, Oral Neoplasm, Ass-1, mouth tumour, tumor of the tongue, Mouth Neoplasm, phosphorylation, eIF-4F 25 kDa subunit, lip and oral cavity carcinoma, dmTAF[[II]]230, AA408052, neoplasm of oral cavity, tumour of the tongue, oral cavity tumour, Project, fold, Analysis, average, increased, PKC-delta, AI385711, TFIID TAF250, TONGUE NEOPL, Analyses, EIF4EL1, cel, neoplasm (disease), tumour of tongue, rapamycin and nutrient-sensitive TOR complex, proteins, Histories, Phosphoprotein, CA, tongue tumor, ASS, historical notes., Mouth Cancers, malignant neoplasm, papilla, signaling process, tumor of tongue, mouth neoplasm, Mouth Cancer, NEOPL TONGUE, associated, single organism signaling, Tongue Cancers, Tongue, malignancy, protein anabolism, dTAF[[II]]230, anatomical protrusion, protein biosynthetic process, dTORC1, tumor of oral cavity, lamina, TAF200, flanges, prkcd, Normalcy, TAFII-250, TAF250/230, historical aspects, NEOPL ORAL, TAFII250, increased period, organ system cancer, shelf, pkc-delta2, protein formation, oral cavity tumor, Ontology Projects, eIF4A-II, NEOPL MOUTH, interferon type I, TOR complex 1, Smoking, Gene Ontologies, Cancer of Mouth, Ontology, tumour of oral cavity, tongue neoplasm, PKCdelta, Ontologies, IFN type I, Projects, shelves, nutrient sensitive complex, Cancer of the Tongue, initiation, Mouth, exposed, CG17603, projection, TAF[[II]], AUTS19, ridge, dTOR/dRaptor complex, Health, npkc-delta, Taf250, protein synthesis, Patient, spine, SR3-5, Mouth Neoplasms, CVID9, cancer, time, accessory, TAF230, hyperphosphorylated, oral cavity neoplasm, TORC1, d230, biological signaling, mRNA cap-binding protein, Smoking Behavior, lamellae, Aspect, cell type cancer, Neoplasms, number, tumour of mouth, Gene, dTAFII250, mouth tumor, slow time, EfW1, presence, process of organ, supernumerary, Oral Neoplasms, Gene Ontology Project, Gene Ontology, protrusion, period, lamella, dmTAF1, Taf230, Gene Products, ALPS3, tongue neoplasm (disease), neoplasm, lip and oral cavity cancer, Cancer of Tongue, malignant tumour, TAF250, BM-010, Normalities, study, Taf200, network topology analysis, dTAF[[II]]250, Oral Cancer, Oral, oral carcinoma, cell, Taf1p, ridges, EIF4GI, EIF-4G1, may1, D14Ertd420e, dTAF250, extruding from, Smoking Habits, Clients, MOUTH NEOPL, Oral Cavity Neoplasm, Habit, Chronic, Behaviors, TAF, laminae, Cancer, eIF-4E, TAF[[II]]250, P220, TORC 1 complex, Tongue Cancer, malignant, exits through, anatomical process, Proteins, Phosphorylations, l(3)84Ab, BG:DS00004.13, Pkcd, Historical Aspects, increased time, Behavior, Client, Historical Aspect, chronic, Cell, oral cancer, dTAF230, development, count in organism, MT, Phosphopeptide, Oral Cancers, pkcd, PKC[d], p230, malignant neoplasm (disease), Smoking Habit, chemical analysis, Protein, MAY1, PARK18, high time, Neoplasm, Tongue Neoplasms, TAF[[II]]250/230, TFIID, protein biosynthesis, prolonged period, PKCD, flange, organ process, mTORC1, EIF4E1, oral cavity neoplasm (disease), neoplasm of the tongue, Ontology Project, Taf[[II]]250, EIF4A, primary cancer, TAF[[II]]230, Normality, increased number, CBP, tumor of mouth, TAF[II]250, Cancers, Keratinocyte, EIF4F, EIF4G, malignant tumor, EIF4E, signalling, Protein Gene Products, Gene Proteins, processes, process, present in greater numbers in organism, eIF4E, DmelCG17603, signalling process, malignant neoplastic disease, Data, tongue tumour, Smoking Behaviors, processus, assay, quantitative, DDX2B, Aspects, eIF-4A-II, Data Analyses, Habits, Historical, presence or absence in organism, TAF1ORAL NEOPL, projections, protein translation, Cancer of the Mouth, neoplasm of tongue, Oral Neoplasm, determination, Ass-1, mouth tumour, tumor of the tongue, Mouth Neoplasm, extracted material, phosphorylation, eIF-4F 25 kDa subunit, lip and oral cavity carcinoma, dmTAF[[II]]230, peptide, Polypeptides, peptido, neoplasm of oral cavity, AA408052, tumour of the tongue, oral cavity tumour, fold, Analysis, increased, TONGUE NEOPL, TFIID TAF250, peptides, Analyses, EIF4EL1, cel, tumour of tongue, rapamycin and nutrient-sensitive TOR complex, proteins, Histories, tongue tumor, ASS, historical notes., Mouth Cancers, papilla, signaling process, tumorous, tumor of tongue, mouth neoplasm, Mouth Cancer, NEOPL TONGUE, associated, Tongue Cancers, single organism signaling, Tongue, protein anabolism, dTAF[[II]]230, anatomical protrusion, protein biosynthetic process, dTORC1, tumor of oral cavity, lamina, TAF200, flanges, TAFII-250, TAF250/230, historical aspects, NEOPL ORAL, TAFII250, increased period, shelf, protein formation, oral cavity tumor, eIF4A-II, NEOPL MOUTH, interferon type I, TOR complex 1, Smoking, Cancer of Mouth, tumour of oral cavity, tongue neoplasm, IFN type I, shelves, nutrient sensitive complex, Cancer of the Tongue, Mouth, CG17603, projection, TAF[[II]], AUTS19, ridge, dTOR/dRaptor complex, Taf250, protein synthesis, Patient, spine, SR3-5, Mouth Neoplasms, Polypeptide, time, accessory, TAF230, oral cavity neoplasm, TORC1, d230, biological signaling, mRNA cap-binding protein, Smoking Behavior, lamellae, Aspect, Neoplasms, tumour of mouth, number, Gene, mouth tumor, dTAFII250, slow time, EfW1, Oral Neoplasms, presence, process of organ, supernumerary, protrusion, period, lamella, dmTAF1, Taf230, Gene Products, tongue neoplasm (disease), lip and oral cavity cancer, Cancer of Tongue, TAF250, BM-010, Taf200, network topology analysis, dTAF[[II]]250, Oral Cancer, Oral, oral carcinoma, cell, Taf1p, ridges, EIF4GI, EIF-4G1, dTAF250, Smoking Habits, Clients, MOUTH NEOPL, Oral Cavity Neoplasm, Habit, Chronic, peptidos, Behaviors, TAF, laminae, Cancer, eIF-4E, TAF[[II]]250, P220, Tongue Cancer, TORC 1 complex, anatomical process, Proteins, Phosphorylations, l(3)84Ab, BG:DS00004.13, Historical Aspects, increased time, Behavior, Client, Historical Aspect, chronic, Cell, Peptide, oral cancer, dTAF230, polypeptide, count in organism, Oral Cancers, p230, Smoking Habit, chemical analysis, Protein, PARK18, Neoplasm, Tongue Neoplasms, high time, TAF[[II]]250/230, TFIID, protein biosynthesis, prolonged period, flange, organ process, mTORC1, EIF4E1, oral cavity neoplasm (disease), neoplasm of the tongue, Taf[[II]]250, EIF4A, TAF[[II]]230, increased number, CBP, tumor of mouth, TAF[II]250, Cancers, Keratinocyte, EIF4F, EIF4G, EIF4E, signalling, Protein Gene Products, processes, process, present in greater numbers in organism, Gene Proteins, eIF4E, DmelCG17603, signalling process, Data, tongue tumour, Smoking Behaviors, processus, Peptid, assay, quantitative, DDX2B, Aspects, eIF-4A-II, Data Analyses, Habits, Historical, presence or absence in organism, TAF1high time, Chronic, slow time, prolonged period, increased period, increased time, chronic, Keratinocyte.falseChronic shisha exposure alters phosphoproteome of oral keratinocytesShisha smoking has been epidemiologically linked to development of oral lesions as well as various cancers. However, few molecular studies investigate the pathobiology of shisha induced cancer. In this study we investigate the effects of chronic shisha exposure in an in vitro model using normal, immortalized, non-transformed oral keratinocytes. Quantitative proteomic and phosphoproteomic analyses were performed on OKF6/TERT1 cells exposed to shisha for a period of 8 months. Gene Ontology and pathway analysis of dysregulated proteins and phosphoproteins were carried out to identify significantly enriched biological processes and pathways in shisha exposed cells compared to parental cells. Chronic shisha exposure resulted in increased cell scattering phenomenon in OKF6/TERT1 cells. Quantitative data analysis revealed differential phosphorylation of 164 phosphopeptides (fold change ≥1.5, p≤0.05) corresponding to 136 proteins. Kinases such as PRKCD were seen to be hyperphosphorylated. Pathway analysis revealed an enrichment of hyperphosphorylated and/or overexpressed proteins involved in the mTORC1 and EIF4F complexes. These complexes are associated with initiation of protein translation and are known to the affected in cancers. Network analysis also highlighted a downregulation of proteins associated with Type I interferon signaling in shisha exposed cells. High throughput phosphoproteomic analysis revealed global perturbations to the molecular milieu of oral keratinocytes upon shisha exposure. Further studies are needed to validate putative targets in oral cancer patients with shisha smoking 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