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Proteomic Analysis of a Noninvasive Human Model of Acute Proteomic analysis of a noninvasive human model of acute inflammation and its resolution: the twenty- one day gingivitis model Grant, Melissa; Creese, Andrew; Barr, G; Ling, Martin; Scott, AE; Matthews, John; Griffiths, Helen; Cooper, Helen; Chapple, Iain DOI: 10.1021/pr100446f Citation for published version (Harvard): Grant, M, Creese, A, Barr, G, Ling, M, Scott, AE, Matthews, J, Griffiths, H, Cooper, H & Chapple, I 2010, 'Proteomic analysis of a noninvasive human model of acute inflammation and its resolution: the twenty-one day gingivitis model', Journal of Proteome Research, vol. 9, no. 9, pp. 4732-44. https://doi.org/10.1021/pr100446f Link to publication on Research at Birmingham portal General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. When citing, please reference the published version. Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact [email protected] providing details and we will remove access to the work immediately and investigate. Download date: 01. Feb. 2019 Proteomic Analysis of a Noninvasive Human Model of Acute Inflammation and Its Resolution: The Twenty-one Day Gingivitis Model Melissa M. Grant,*,† Andrew J. Creese,† Gordon Barr,‡ Martin R. Ling,† Ann E. Scott,§ John B. Matthews,† Helen R. Griffiths,| Helen J. Cooper,⊥ and Iain L. C. Chapple† Periodontal Research Group, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St. Chads Queensway, Birmingham, B4 6NN, United Kingdom, WestCHEM, Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, United Kingdom, Unilever Oral Care U.K., Port Sunlight, Quarry Road East, Bebington CH63 3JW, United Kingdom, Life and Health Sciences, Aston University, Birmingham B4 7ET, United Kingdom, and School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom Received May 13, 2010 The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins. Keywords: FTICR-MS • FT-ICR • FT-MS • Fourier transform ion cyclotron mass spectrometry • proteomics • gingivitis • inflammation • iTRAQ • cluster analysis • cilia • ribbon synapses Introduction The model involves periodontally and systemically healthy volunteers, who do not brush specified test teeth in one sextant The 21-day experimental gingivitis model is an established of the dentition, but continue to brush the remaining control teeth noninvasive model in humans for investigating the induction over a 21 day period. Accidental brushing of test teeth is prevented and resolution of inflammation in response to increasing by the use of a vinyl shield (mouth guard) to cover them during bacterial accumulation. From its inception in 1965,1,2 this cleaning. On day 21, the accumulated plaque on all teeth is model of inflammation has been used to assess how drugs,3 removed by a trained hygienist and study participants resume bioactive compounds in dentifrices4-6 and environmental their normal oral hygiene practices. Clinical assessment of plaque accumulation and gingival inflammation are made at baseline, toxins, such as tobacco smoke,7,8 affect the development of day 7, 14, 21 on test and control teeth, following plaque ac- inflammation. The model is designed to enable the study of cumulation on test teeth, and normal plaque removal from both the induction and resolution of inflammation, which can control teeth. Assessments are repeated again on day 35, following also be assessed over a relatively short period of time (35 days) 14 days of resolution of inflammation at test sites, and mainte- and in a controlled manner. nance of health at control sites. When performed by trained and calibrated experts, these measures show consistent increases in * To whom correspondence should be addressed. E-mail: m.m.grant@ bham.ac.uk. plaque-induced inflammation up to day 21 followed by a return † College of Medical and Dental Sciences, University of Birmingham. to baseline, preinflammation levels at day 35.9,10 However none ‡ University of Glasgow. of these assessments could potentially form the basis for a § Unilever Oral Care U.K. | Aston University. predictor of disease as they are all measures of existing inflam- ⊥ College of Life and Environmental Sciences, University of Birmingham. mation or inducers of inflammation. 4732 Journal of Proteome Research 2010, 9, 4732–4744 10.1021/pr100446f 2010 American Chemical Society Published on Web 07/27/2010 Acute Inflammation and Its Resolution research articles A further and more objective assessment that can be made fragmented typically by collision induced dissociation31 (CID) during the experimental gingivitis is that of the volume and resulting in b and y ion series.32 The MS/MS spectra are composition of gingival crevicular fluid (GCF) and at individual searched against protein databases by use of algorithms (e.g., sites around individual teeth. This fluid flows from the crevice Mascot,33 SEQUEST,34 OMSSA35) which match the data to between the tooth and the gum and comprises both a serum theoretical spectra from in-silico digests of proteins. Quantita- transdate11 and tissue exudates.3,10,12-14 During inflammation, tive proteomics by mass spectrometry can use several methods. the volume and flow of this fluid increases,1 and this alone can Two of the most popular are stable isotope labeled amino acids be used to demonstrate induction and/or presence of gingivitis in cell culture36 (SILAC) and isobaric tags for relative and and periodontitis. However, due to its very nature, as a mixed absolute quantitation (iTRAQ).37 In SILAC, cells are grown in serum transudate and tissue exudate, GCF carries with it media lacking essential amino acids. The media is supple- proteins from the crevice. Thus its composition also provides mented with heavy or light amino acids. The labeled and a biological and pathological fingerprint of the various physi- unlabeled peptides elute together and the intensity of the ological and biochemical processes occurring within the gin- peptide ions can be compared. SILAC is not suitable for clinical gival tissues. In 1980, Novaes et al.15 demonstrated that the samples. A common approach for quantitation of biological total protein present in GCF increased with increasing severity fluids38 and tissue samples39 is postdigestion labeling with of periodontitis, although this was probably due to increases iTRAQ40 labels. iTRAQ labels consist of a reporter ion with a in GCF volume. By 1985, Lamster et al.16 had measured m/z value of between 113 and 121 and a balance mass differences in lactate dehydrogenase, beta-glucuronidase and (191-183 Da) such that all labeled peptides of the same arylsulfatase activity in GCF collected from experimental sequence have the same nominal mass shift (∆304 Da). Test gingivitis sites and demonstrated increases in these biomarkers and control samples are treated with separate iTRAQ labels over the course of the 21 day study. Since then, studies have postdigestion and combined prior to MS analysis. When the targeted particular cytokines,3,10,12-14,17,18 antibacterial peptides peptide ions are fragmented with CID, the reporter
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