Proteomic Analysis of Gingival Tissue and Alveolar Bone during Alveolar Bone Healing

• DOI: 10.1074/mcp.m112.026740
• Publication Date: 2013-07-04T06:41:36Z
• Authors: Hee-Young Yang; Joseph Kwon; Min-Suk Kook; Seong Soo Kang; Se Eun Kim; Sungoh Sohn; Seunggon Jung; Sang-Oh Kwon; Hyung-Seok Kim; Jae Hyuk Lee; Tae-Hoon Lee
• Keywords: Bone tissue; Bone remodeling

Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves build-up of osteogenic cells, which orchestrate remodeling/healing through expression numerous mediators signaling molecules. Periodontal models have proven useful for studying interaction communication between alveolar bone soft tissue. We applied a quantitative proteomic approach to analyze compare proteins with altered in gingival following tooth extraction. For target identification validation, hard were extracted from mini-pigs at indicated times after From triplicate experiments, 56 27 found be differentially expressed before The 21 those was both bone. Comparison activated networks highlighted their distinct responsibilities healing. Moreover, we that there crosstalk identified respect cellular assembly, organization, communication. Among these proteins, examined detail patterns associated ATP5B fibronectin 1. involved nucleic acid metabolism, small molecule biochemistry, neurological disease, 1 maintenance. Collectively, our findings indicate accompanied profound among regulating resources, they provide novel insight into molecular mechanisms healing periodontal Healthy dental surrounds teeth are essential proper function teeth, as well good appearance general health. Socket extraction experimental model investigating Preservation socket requires formation biological connection living osseous tissue, has created during process. success such remodeling dependent on establishment barrier able shelter underlying structures osseo-integration Understanding processes governing maintenance around paramount oral Several studies reported significant structural changes reabsorption fresh sockets extraction, important dimensional (1Araujo M.G. Lindhe J. Dimensional ridge alterations An study dog.J. Clin. 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Shetty S.K. autologous degree defects.J. 2009; 36: 581-588Crossref (52) platelet-derived growth factor (11Nevins Camelo M.L. Schenk R.K. Lynch S.E. humans recombinant factor-BB (rhPDGF-BB) allogenic bone.J. 1282-1292Crossref (277) 12Camelo Class furcations purified allograft.Int. 23: 213-225PubMed morphogenic (BMPs) 1The abbreviations used are:ATP5BATP synthase subunit beta, mitochondrialBGNbiglycanBMPbone morphogenetic proteinDCNdecorinECMextracellular matrixFN1fibronectin 1MSEhigh/low-collision-energy mass spectrometrySERPINF1serpin peptidase inhibitor, clade F, member 1TGF-βtransforming βUPLCultraperformance liquid chromatography. Scholar) promises potentially better outcomes treatments, although efficacy remains controversial. ATP mitochondrial biglycan protein decorin extracellular high/low-collision-energy spectrometry serpin transforming β ultraperformance all life ongoing bone, most final products homeostatic pathways. Profiling vital thorough understanding biology. To date, proteome research focused mainly vitro analysis bone-forming cells (osteoblasts osteoclasts) determine under given set conditions (13Kubota Wakabayashi Matsuoka Proteome secreted osteoclast differentiation two different methods: two-dimensional electrophoresis isotope-coded affinity tags chromatography.Proteomics. 3: 616-626Crossref (77) 14Wang Park J.S. Chu Krakowski Luo D.J. Proteomic profiling marrow mesenchymal stem upon beta1 stimulation.J. Biol. Chem. 279: 43725-43734Abstract (219) 15Chang E.J. Kwak H.B. Kim J.C. Lee Z.H. H.H. Elucidation CPX-1 RANKL-induced osteoclastogenesis proteomics approach.FEBS Lett. 564: 166-170Crossref (19) 16Salasznyk R.M. Westcott A.M. Klees R.F. Ward D.F. Xiang Vandenberg Bennett Plopper G.E. Comparing profiles osteoblasts gene ontologies.Stem Cells Dev. 14: 354-366Crossref (58) important, cannot identify actual profile Recently, directly skull (17Fan Liu Wang Shi F. Xiong L. He Peng Functional bones rats osteoporosis ovariectomy.Life Sci. 76: 2893-2901Crossref (25) 18Pastorelli R. Carpi Airoldi Chiabrando Bagnati Fanelli Moverare Ohlsson vivo estrogen-regulated bone.Proteomics. 5: 4936-4945Crossref (39) first separated gel electrophoresis, spots interest excised via (MS). extreme (e.g. extremely basic or acidic, large, hydrophobic) challenging. Shotgun proteomics, method high-throughput (19Aebersold Mann Mass spectrometry-based proteomics.Nature. 422: 198-207Crossref (5585) 20Xie Ye Jiang Jin Zou Octadecylated silica monolith capillary column integrated nanoelectrospray ionization emitter highly efficient analysis.Mol. Cell. Proteomics. 454-461Abstract (82) 21Peng Elias J.E. Thoreen C.C. Licklider L.J. Gygi S.P. Evaluation multidimensional chromatography coupled tandem (LC/LC-MS/MS) large-scale analysis: yeast proteome.J. Res. 2: 43-50Crossref (1378) avoids intrinsic limitations electrophoresis. Despite an interesting need characterization proteome, one apply shotgun rat femur (22Schreiweis M.A. Butler Kulkarni N.H. Knierman M.D. Higgs R.E. Halladay D.L. Onyia Hale adult reveals presence cartilage/chondrocyte markers.J. Biochem. 2007; 101: 466-476Crossref (29) only 133 because analyzed one-step without demineralization stage. other report showed beagle carried step (23Jiang Feng Cui Method development analysis.J. 6: 2287-2294Crossref (92) There no reports regarding yet. critical issue (24Saravanan R.S. Rose J.K. sample enhanced recalcitrant plant tissues.Proteomics. 4: 2522-2532Crossref (372) Because largely mineralized, therefore nearly solid, classical methods appropriate It necessary develop efficiently extract In earlier analyses 22Schreiweis ground powder, means incubating powder lysis buffer. mechanically breaking down laborious, large animal bones. More amounts collagen proteoglycans also extracted, this impair detection low-abundance strongly affect isoelectric focusing (25Hermansson Sawaji Bolton Alexander Wallace Begum Wait Saklatvala articular cartilage shows increased type synthesis osteoarthritis inhibin betaA (activin A), regulatory chondrocytes.J. 43514-43521Abstract (135) study, adopted alternative demineralizing then investigated efficiency This based recently sequential protocol comprehensive its proteome. Two-dimensional high-performance chromatography–tandem (LC-MS/MS) extracts, enabling 2479 employed similar Given number likely tissues, another goal examine putative Here, nano-UPLC-MSE-based label-free adjacent environment would expected specific regeneration. suggest determining will enable improvement interface, aspects therapy. All chemicals purchased Sigma-Aldrich, Merck, USB (Affymetrix, Inc., Santa Clara, CA). Mini-pigs (miniature pig; PWG Genetics Korea, Ltd) maintained specific-pathogen-free conditions. animal-related procedures reviewed approved Animal Care Regulations Chonnam National University (accession number: CNU IACUC YB-2011-3). analysis, nine pigs subdivided three groups (n = 3 each group) sacrificed week 2 weeks respectively. After mini-pig sacrificed, fragments harvested area (the fourth premolars right left sides maxillary regions). trimmed separate Both stored −70 °C until analyzed. homogenized buffer containing 1% Triton X-100, 20 mm Tris-HCl (pH 7.5), 150 NaCl, EDTA, EGTA, 2.5 sodium pyrophosphate, β-glycerolphosphate, orthovanadate, 25 fluoride, μg/ml leupeptin, PMSF. over 4-h period 4 °C, sonicated cleared centrifugation 13,000 rpm 30 min °C. concentration measured BCA assay (Thermo Scientific, Rockford, IL), samples 200 μg subjected 10% SDS-PAGE. visualized staining Coomassie Blue R-250, gels washed distilled water prior tryptic digestion. trypsinization, collected excising 10 slices sample, described previously (26Yang H.Y. Kwon Cho Choi H.I. H.R. S.H. Chung K.J. Ryoo Z.Y. K.O. T.H. affected peroxiredoxin V knock-down hypoxic kidney.J. 2010; 4003-4015Crossref chopped 1-mm3 pieces destained overnight incubation 50% acetonitrile 50 ammonium bicarbonate. Each piece reduced dithiothreitol alkylated 55 N-ethylmaleimide 100 Following digestion (2 μg/sliced gel; Promega, Madison, WI) 16 h 37 peptides recovered sliced 5% formic acetonitrile. dried vacuum centrifuge combined single tube. desalted solid-phase Oasis HLB C18 microelution plate (Waters, Milford, MA) −80 nano-LC-MS/MS comparative proteomics. Protein et al., some modifications (Fig. 1A) Alveolar (about long × wide 10−20 thick) nitrogen incubated PBS 7.4) protease inhibitor mixture remove contaminants. Thereafter, about mg 1.2 m HCl demineralize (200 slice per milliliter solution). centrifugation, supernatant Extract pellet Tris, 6 guanidine-HCl 7.4), 72 2. further solution (100 guanidine-HCl, pH 0.5 tetrasodium EDTA centrifugation. Finally, remaining centrifugations out 12,000 crude extracts precipitated acetone −20 four (Extracts 1–4) separately dissolved μl Tris 8.1). Once solution, (total volume: 800 μl) determined Bradford assays (Bio-Rad Laboratories), 200-μg aliquots Separations performed nano-UPLC RP (75 μm 250 mm; particle size, 1.7 μm) enrichment Symmetry (180 5 nano-ACQUITY Ultra PerformanceTM system (Waters Corporation, MA). LC gradient program operation mode (27Xu Suenaga N. Edelmann Fridman Muschel R.J. Kessler B.M. Novel MMP-9 substrates cancer revealed approach.Mol. 2008; 7: 2215-2228Abstract Trypsin-digested (5 loaded onto mobile phase (3% 0.1% acid), flow rate 300 nl/min. included 3%–40% B (97% acid) 95 40%–70% min, followed sharp increase 80% within min. Sodium formate (1 dissolving NaOH isopropanol 0.2% HCOOH (1:1, v/v) externally calibrate TOF analyzer range m/z 50–2000 series singly doubly charged ion clusters LC-MS/MS experiments (28Moon Y.J. Yun Lim H.L. M.S. Kang S.G. J.H. S.I. Y.H. hydrogen-producing hyperthermophilic archaeon Thermococcus onnurineus NA1 one-carbon substrate culture conditions.Mol. 2012; 11 (M111.015420)Abstract (30) accuracy raw data corrected against monoisotope [Glu1]-fibrinopeptide (m/z 785.8426 Da [M + 2H]2+). fmol/μl, 600 nl/min rate) infused every spectrometer lock correction analysis. During acquisition, collision energy low-energy MS elevated-energy (MSE) 15–40 eV ramping mode, One cycle MSE 3.2 s. cycle, spectra acquired 1.5 s 0.1-s interscan delay 300–1990), ions exceeding counts selected fragmentation cell 50–2000). LC-MSE processed searched ProteinLynx GlobalServer version 2.3.3 Corporation) reconstruct MS/MS combining masses identical retention times. database search Sus scrofa database, retrieved NCBI website 27,041 entries, data. identification, parent tolerance ppm, fragment 0.2 Da. Carbamidomethylation (+57 Da) cysteine methionine oxidation (+16 chosen variable modifications, least allowed if confidence greater than 95% basis IDENTITYE algorithm (21Peng false-positive bank query option automatically generated reversed 2.3.3. ExpressionTM software results exact time table peptide values (supplemental Fig. S2). Analysis abundance, measurements peak intensities observed low-collision-energy (MS) set, (version 2.3.3., Waters Peptides fragmented clustered chemically components together sample. Precursors align deviation threshold (<0.25 min) precision (<15 ppm) normalization dataset, spiked digested standard including alcohol dehydrogenase pmole), rabbit glycogen phosphorylase (0.5 enolase (4 serum albumin (8 pmole) (Mass PREP Digestion Standard Mixture, No. 186002866, Waters) internal standard. Yeast datasets. Ingenuity Pathway (IPA 8.0, Systems Redmond City, CA) knowledge-based network canonical pathway nano-LC-MSE built-in function. STRING 9.0 queried symbols supplemental S1. parameters medium (0.400) prediction options "Co-expression," "Experiments," "Databases." Total RNA isolated porcine (3 h, week, extraction) QIAzol® Lysis reagent (Qiagen Sciences, Valencia, CA), cDNAs synthesized PrimeScript™ RT Reagent Kit real-time PCR (Takara Biotechnology, Japan) according manufacturer's instructions. Quantitative ABI 7300 Prism SDS (Applied Biosystems, Foster SYBR® Premix Ex Tag kit Biotechnology) temperature protocol. obtained relative quantities calculated 2-ΔΔCT (expressed fold ratio). Hypoxanthine phosphoribosyltransferase control normalization, performed. Supplemental Table S1 lists primers (Sus Scrofa) PCR. Samples (30 μg) gingiva (soft tissues) 12% polyacrylamide gels. transferred polyvinylidene fluoride anti-ATPB (NBP1