Volume 85 • Number 6
Proteomic Analyses of Human Gingival and Periodontal Ligament Fibroblasts Holly McKnight,* W. Patrick Kelsey,* Deborah A. Hooper,* Thomas C. Hart,† and Angelo Mariotti*
Background: Although human gingival fibroblasts (hGFs) and human periodontal ligament fibroblasts (hPDLFs) exhibit numerous phenotypic similarities, it has been suggested that the secretory and behavioral differences, which exist between these cell types, are a result of the membrane protein composition of these cells. Methods: Four matched pairs of hGFs and hPDLFs were cultured. Before confluence, membrane-bound and -associated proteins from cells of the fourth passage were extracted. The processed protein samples were evaluated using capillary-liquid chromatography-nanospray tandem mass spectrometry. Global protein identification was performed on an orbitrap mass spectrometer equipped with a microspray source operated in positive ion mode. Proteome software was used to validate protein identifications derived from tandem mass spectrometry sequencing results. Results: Four hundred fifty proteins were common to both hGFs and hPDLFs. Of the proteins identified, 214 were known membrane-bound or -associated proteins, and 165 proteins were known nuclear-associated proteins. Twenty-seven proteins, identified from the 450 proteins, common to both hGFs and hPDLFs, were detected in statistically significant greater quantities in either hGFs or hPDLFs. More specifically, 13 proteins were detected in significantly greater quantities in hGFs, whereas 14 proteins were detected in significantly greater quantities in hPDLFs. Conclusions: Distinct differences in the cellular protein catalog may reflect the dynamic role and high energy requirements of hGFs in extracellular matrix remodeling and response to inflammatory challenge as well as the role of hPDLFs in monitoring mechanical stress and maintaining tissue homeostasis during regeneration and remineralization. J Periodontol 2014;85:810-818. KEY WORDS Fibroblasts; gingiva; periodontal ligament; proteome. * Division of Periodontology, College of Dentistry, The Ohio State University, Columbus, OH. † Department of Periodontics, College of Dentistry, University of Illinois at Chicago, Chicago, IL.
O
nce thought to be static cells of connective tissues (CTs), fibroblasts have reemerged as dynamic cells responsible for the health, function, and immunity of a tissue. Human fibroblasts demonstrate selfrenewal capacity and multipotent potential, indicating their ability to perform a range of cellular functions beyond basic homeostasis that includes both repair and regeneration.1,2 Because fibroblast attachment to the extracellular matrix (ECM) helps to maintain cell shape and function in addition to tissue integrity, it has been suggested that fibroblast behavior is essentially controlled by a balance of the synthesis and degradation of the ECM.3,4 Although found in many different tissues, fibroblasts have functional characteristics that are unique to their biologic niche. For example, proliferation, mobility, production of ECM, ability to respond to microbial and viral challenges, metabolism and repair of the ECM, and regenerative capacity will vary depending on the tissue source of the fibroblast.5 Considering the importance of the periodontal ligament (PDL) in the maintenance and regeneration of the periodontium, defining cellular behavior of the principal resident of this tissue, the fibroblast, is essential to understanding regulation of the periodontium. Although human gingival fibroblasts (hGFs) and human PDL fibroblasts (hPDLFs) exhibit numerous similarities, it has been posited that secretory and behavioral differences exist between these cell types. From an embryologic
doi: 10.1902/jop.2013.130161
810
J Periodontol • June 2014
viewpoint, hGFs and hPDLFs do not originate from the same tissues. hPDLFs are derived from the inner layer of the dental follicle and are ectomesenchymal in origin.6 Derivation of hGFs is not completely understood, but it is thought that these cells develop from either the dental follicle or embryonic ridge mucosa and are mesenchymal cells.7 In addition to their embryologic origin, hGFs and hPDLFs have been noted in cell culture to differ in proliferative capacity and glycosaminoglycan content,8 chemotactic response,9 apoptotic propensity,10 protein and collagen synthesis,11 as well as the capacity to form mineralized tissue.12 Gene expression profiles were reported to differ between hGFs and hPDLFs, reflecting intrinsic functional differences of the two cell populations and fundamental differences in their tissue-specific activities.10 Because genes affect protein levels and functional differences in cells are manifest at the protein level, functional differences between hGFs and hPDLFs may derive from differences of their respective proteomes. In dentistry, research on the proteome has focused on oral flora and pathogens, salivary secretions, and protein expression of hard tissues. Proteomic biomarkers in caries pathogen profiles have been found to be useful in predicting caries in a pediatric population.13 In endodontic lesions, proteomics has been used to demonstrate the complexity of microbiota involved in the pathogenesis of the necrotic pulp.14 Proteomic analysis of dental hard tissues has been used to describe the protein expression of dentin to define the organic matrix of dentin and to characterize odontoblasts.15 In a review of published studies regarding proteomes of oral soft tissues, only three articles pertain to the human gingiva or periodontium.16-18 These studies are broad protein surveys of the PDL,16 neutrophils of patients with aggressive periodontitis,17 and/or drug-associated enlargement of gingival tissues.18 Unlike the broad surveys of proteins in tissues or cells, the purpose of this study focuses on membrane-bound and membraneassociated proteins found in either hGFs or hPDLFs. Although membrane-bound proteins are difficult to isolate, their influence on cell function is significant, because these are the proteins through which cells monitor their environments, maintain homeostasis, and signal changes to the nucleus that ultimately affect cellular and biologic responses to the monitored environment.19,20 Membrane-bound and/or -associated proteins not only are necessary for numerous cellular functions, changes or disruptions to them drive signal transduction, substantially affecting tissue function and the health of the individual. For example, disruptions in nuclear lamina,21 an essential element of the nuclear envelope, have been implicated in Emery-Drifuss muscular dystrophy, autosomal
McKnight, Kelsey, Hooper, Hart, Mariotti
recessive Charcot-Marie-Tooth disorder type 2B1, and autosomal recessive manibuloacral dysplasia.22 Considering how important membrane proteins are for cellular function, this investigation was designed to evaluate the putative differences in the membrane-bound and -associated proteome of hGFs and hPDLFs. MATERIALS AND METHODS Collection of Samples Fibroblasts were harvested from human gingiva and hPDLs of individuals with planned extraction of impacted third molars at the Division of Oral and Maxillofacial Surgery at The Ohio State University College of Dentistry. Teeth included for the harvest of PDLFs had three-fourths or more root formation and were completely or partially bony impacted with radiographic evidence of a dental follicle present and lack of communication with the oral cavity. Potential participants had a diagnosis of gingival health, which was confirmed by the absence of gingival inflammation or infection. Interested participants who met inclusion criteria provided signature of informed consent. This study was approved by the Institutional Review Board of The Ohio State University. Inclusion criteria for this study were as follows: 1) healthy patients without a history of systemic disease; 2) aged 16 years or older; 3) a patient of record of The Ohio State University College of Dentistry; and 4) a previously signed treatment plan for extraction of third molar(s). Tissues from four individuals (one male and three females, aged 16 to 36 years; mean age: 20.1 years) who consented to participate were used. Culture of Fibroblasts Four matched pairs of hGFs and hPDLFs were cultured as described previously.23 Briefly, hGFs were harvested from the gingiva of the overlying flap of the impacted third molars. All gingival samples appeared clinically healthy (firm, non-edematous, and coral pink) at biopsy. hPDLFs were harvested from the middle third of the root of impacted third molars. At the time of sampling, explants were immediately placed in 10 mL biopsy media, which consisted of Eagle minimal essential medium supplemented with 10% fetal bovine serum, 350 mg/mL L-glutamine, 100 mg/mL penicillin, and 100 mg/mL streptomycin. Fibroblasts were propagated in 25-cm2 flasks in media at 37C in a humidified incubator with 5% CO2 and 95% air atmosphere. Media were exchanged in the flasks every 48 hours. All experiments were performed only on cells in the fourth passage. Isolation and Solubilization of Membrane-Bound and -Associated Proteins Before confluence, enrichment of membrane-bound and -associated proteins from cells of the fourth 811
Periodontal Cell Proteome
passage was undertaken according to the modified protocol of Chevallet et al.24 Briefly, cell pellets were suspended in 40 mM Tris-HCl (pH 8.0) with protease inhibitor at a concentration of 200 mL/1 mL A600 pellet size and transferred to a 1.5-mL microcentrifuge tube for cell fraction by sonication on ice with medium power for three cycles of 10 seconds. After fractionation, the cell lysate was centrifuged for 15 minutes at 4C at 10,000 rpm to pellet insoluble cellular components. The pellet of the insoluble proteins was resuspended in 200 mL lysis buffer consisting of 40 mM Tris-HCl (pH 8.0), 7 M urea, 2 M thiourea, 0.25% weight-to-volume amidosulfobetaine-14, and 0.25% of 100% Nonidet-40 and incubated at 22C for 30 minutes. Subsequently, samples were mixed for 1 minute and centrifuged (13,200 rpm) for 30 minutes at 22C. The supernatant, containing the membrane proteins with some lipid, nucleic acid, and sugar components, was diluted to 500 mL with distilled water. Twenty percent of fresh trichloroacetic acid was added to suspension and incubated on ice for 1 hour to remove contaminants and centrifuged (13,200 rpm) for 15 minutes. The pellet was recovered, and this procedure was repeated. Without disturbing the pellet, 10 mL supersaturated Tris base was added to the pellet. After 5 minutes of incubation, 1 mL iced acetone was added to the pellet and mixed, and the protein suspension was centrifuged (13,200 rpm) for 15 minutes at 4C. The acetone was removed, and the pellet was allowed to air dry and resuspended in 2 mL/200 mL protein solubilizer‡ and 2 M urea. Peptide Separation, Protein Identification, and Quantitation The processed protein samples were submitted for enzyme digestion and identification at the Mass Spectrometry and Proteomics Facility at The Ohio State University for label-free proteomic quantitation methodology. Briefly, protein was reduced by adding 10 mL dithiothreitol (5 mg/mL solution prepared in 100 mM ammonium bicarbonate), and the reaction proceeded at 60C for 30 minutes. To block reduced cysteine residues, 10 mL iodoacetamide solution (15 mg/mL in 100 mM ammonium bicarbonate) was added to the sample and allowed to incubate at room temperature for 15 minutes in the dark. Trypsin was prepared in 50 mM ammonium bicarbonate and added to the protein solution with an enzyme to substrate ratio of 1:25 (weight-to-volume). The sample was incubated for 2 hours at 37C before being quenched by acidification. Capillary-liquid chromatography-nanospray tandem mass spectrometry (LC-MS/MS) of global protein identification was performed on an orbitrap mass 812
Volume 85 • Number 6
spectrometer§ equipped with a microspray source operating in positive ion mode. Samples were separated on a capillary columnii using a high-performance liquid chromatography system.¶ Each sample was injected into the precolumn cartridge# and desalted with 50 mM acetic acid for 10 minutes. After injection, the peptides were eluted off of the trap onto the column. Mobile phase A was 0.1% formic acid in water, and 0.1% formic acid in acetonitrile was used as mobile phase B. Flow rate was set at 2 mL/minute. Typically, mobile phase B was increased from 2% to 50% in 90 to 250 minutes, depending on the complexity of the sample, to separate the peptides. Mobile phase B was then increased from 50% to 90% in 5 minutes and kept at 90% for another 5 minutes before being brought back quickly to 2% in 1 minute. The column was equilibrated at 2% of mobile phase B (or 98% mobile phase A) for 30 minutes before the next sample injection. MS/MS data were acquired with a spray voltage of 2 kV at a capillary temperature of 175C. The scan sequence of the mass spectrometer was based on the data-dependent TopTen method: the analysis was programmed for a full scan recorded from 300 to 2,000 Da and a MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive scans of the 10 most abundant peaks in the spectrum. The resolution of the full scan was set at 30,000 to achieve high mass accuracy MS determination. The collusion-induced dissociation fragmentation energy was set to 35%. Dynamic exclusion is enabled with a repeat count of 30 seconds, exclusion duration of 350 seconds, a low mass width of 0.50 Da, and high mass width of 1.50 Da. Multiple MS/MS detection of the same peptide was excluded after detecting it three times. Sequence information from the MS/MS data was processed by converting the raw (.raw) files into a merged file (.mgf) using an in-house program.** The resulting .mgf files were searched,†† and the database was searched against protein sequence databases.25,26 The mass accuracy of the precursor ions was set to 2.0 Da given that the data were acquired on an ion trap mass analyzer, and the fragment mass accuracy was set to 0.5 Da. Considered modifications were methionine oxidation and carbamidomethyl cysteine. A proteome software‡‡27 ‡
Invitrosol protein solublizer, Invitrogen, Thermo Fisher Scientific, Waltham, MA. § Finnigan LTQ orbitrap mass spectrometer, Thermo Fisher Scientific. ˚ , Michrom Bioresources, Auburn, ii 0.2 · 150 mm Magic C18AQ 3m 200A CA. ¶ UltiMate 3000 HPLC system, Thermo Fisher Scientific. # Thermo Scientific Scientific. ** RAW2MZXML_n_MGF_batch (merge.pl, a Perl script). Maintained by the Mass Spectrometry and Proteomics Facility at The Ohio State University, Columbus, Ohio. †† Mascot Daemon v.2.3.2, Matrix Science, Boston, MA. ‡‡ Scaffold, Proteome Software, Portland, OR.
J Periodontol • June 2014
was used to validate protein identifications derived from MS/MS sequencing results. The software verifies peptide identifications assigned by mass spectrometry data analysis programs§§iiii and then probabilistically validates these peptide identifications and derives corresponding protein probabilities. Protein Function and Ontology Protein ontology was assessed using literature reports of protein functions as well as Web-based tools28,29 to identify functional annotation to characterize molecular functions and biologic processes that were used in reporting the potential relationships and interconnecting ontologies for proteins determined to show differential expression in hGFs and hPDLFs. Statistical Analyses The mean, standard deviation, confidence interval, paired t test, and Fisher exact test for all proteins were determined using the datasets of the four matched hGF and hPDLF membrane pellets using bioinformatic software and statistical program analysis by the proteome software. Algorithms for calculating protein probabilities from peptide probabilities were performed using the proteome software. RESULTS Four matched pairs of hGF and hPDLF strains were successfully propagated and underwent protein isolation and proteomic analysis from fibroblasts derived from the overlying gingiva of impacted third molars and the PDL of the impacted third molars. The four source individuals ranged in age from 16.4 to 25.4 years, with a mean age of 20.1 years. Three source individuals were female, and one was male. All participants were systemically healthy with no medication use reported, with the exception of one female, who reported taking an oral contraceptive. All participants were never-smokers. Intraoral examinations determined that all participants were free of any signs or symptoms of gingival inflammation, periodontal disease, or oral infections. A total of 518 proteins were identified from the eight samples analyzed via LC-MS/MS (dataset identifier PXD000387 and DOI 10.6.019/PXD000387).30 Four hundred fifty proteins were common to both hGF and hPDLF. Of the proteins identified, 214 were known membrane-bound or -associated proteins (dataset identifier PXD000387 and DOI 10.6.019/ PXD000387),30 and 165 proteins were known nuclearassociated proteins (dataset identifier PXD000387 and DOI 10.6.019/PXD000387).30 Twenty-seven proteins, identified from the 450 proteins common to both hGF and hPDLF, were detected in statistically significantly greater quantities in either hGFs or hPDLFs (Figs. 1 and 2). Thirteen proteins were detected in significantly greater
McKnight, Kelsey, Hooper, Hart, Mariotti
quantities in hGFs (Figs. 1 and 2). These proteins included aminopeptidase N, A-kinase anchor protein 2, annexin A6, annexin A11, UPF0556 protein C19orf10, prohibitin-2, nicotinamide adenine dinucleotide (NADH)-ubiquinone oxidoreductase 75 kDa mitochondrial, protein AHNAK2, annexin A4, acetylcoenzyme A (CoA) acetyltransferase mitochondrial, microtubule-associated protein 4, Src homology 3 (SH3) domain-binding glutamic acid-rich-like protein 3, and trifunctional enzyme subunit b mitochondrial. Four membrane or associated proteins were detected in greater quantities in hGFs compared with hPDLFs (Fig. 1). These proteins include annexin A4, aminopeptidase N, prohibitin-2, and NADH-ubiquinone oxidoreductase 75 kDa subunit. The three proteins that were detected in greater quantities in the hGFs that were nuclear-associated proteins were annexin A11, protein AHNAK2, and SH3 domain-binding glutamic acid-rich-like protein 3 (Fig. 1). Fourteen proteins were detected in significantly greater quantities in hPDLFs compared with hGFs (Figs. 1 and 2). These proteins include a-2-HSglycoprotein, calcium/calmodulin-dependent protein kinase type II subunit d, desmoplakin, serum albumin, membrane-associated progesterone receptor component 2, basement membrane-specific heparin sulfate proteoglycan core protein, epidermal growth factor (EGF)-like repeat and discoidin I-like domaincontaining protein 3, filaggrin, voltage-dependent calcium channel subunit a-2/d-1, 4F2 cell-surface antigen heavy chain, nucleobindin-1, four and a half LIM domains protein 2, ubiquitin carboxyl-terminal hydrolase isozyme L1, and profilin-1. Nine of the proteins identified preferentially in hPDLFs compared with hGFs were membrane-bound or -associated proteins (Fig. 1). These proteins include calcium/ calmodulin-dependent protein kinase type II subunit d, desmoplakin, voltage-dependent calcium channel subunit a-2/d-1, 4F2 cell-surface antigen heavy chain, nucleobindin-1, membrane-associated progesterone receptor component 2, four and a half LIM domains protein 2, and ubiquitin carboxyl-terminal hydrolase isozyme L1. The remaining three proteins that were detected in greater quantities in the hPDLFs that were nuclear-associated proteins were four and half LIM domains protein 2, calcium/calmodulindependent protein kinase type II subunit d, and ubiquitin carboxyl-terminal hydrolase isozyme L1 (Fig. 1). Forty of the 518 proteins identified by proteomic analysis were only detected in hGFs (Table 1). Of the 40 proteins, 18 were membrane-bound or -associated proteins (Table 1). Twenty-nine of the 518 proteins identified by proteomic analysis were only §§ SEQUEST, Thermo Fisher Scientific. iiii Mascot Daemon v.2.3.2, Matrix Science.
813
Periodontal Cell Proteome
Volume 85 • Number 6
Figure 1.
Membrane/nuclear-associated proteins in the hGFs and hPDLFs. Data are expressed as the mean – SD. All proteins listed exhibit a statistically significant difference (P
Proteomic Analyses of Human Gingival and Periodontal Ligament Fibroblasts Holly McKnight,* W. Patrick Kelsey,* Deborah A. Hooper,* Thomas C. Hart,† and Angelo Mariotti*
Background: Although human gingival fibroblasts (hGFs) and human periodontal ligament fibroblasts (hPDLFs) exhibit numerous phenotypic similarities, it has been suggested that the secretory and behavioral differences, which exist between these cell types, are a result of the membrane protein composition of these cells. Methods: Four matched pairs of hGFs and hPDLFs were cultured. Before confluence, membrane-bound and -associated proteins from cells of the fourth passage were extracted. The processed protein samples were evaluated using capillary-liquid chromatography-nanospray tandem mass spectrometry. Global protein identification was performed on an orbitrap mass spectrometer equipped with a microspray source operated in positive ion mode. Proteome software was used to validate protein identifications derived from tandem mass spectrometry sequencing results. Results: Four hundred fifty proteins were common to both hGFs and hPDLFs. Of the proteins identified, 214 were known membrane-bound or -associated proteins, and 165 proteins were known nuclear-associated proteins. Twenty-seven proteins, identified from the 450 proteins, common to both hGFs and hPDLFs, were detected in statistically significant greater quantities in either hGFs or hPDLFs. More specifically, 13 proteins were detected in significantly greater quantities in hGFs, whereas 14 proteins were detected in significantly greater quantities in hPDLFs. Conclusions: Distinct differences in the cellular protein catalog may reflect the dynamic role and high energy requirements of hGFs in extracellular matrix remodeling and response to inflammatory challenge as well as the role of hPDLFs in monitoring mechanical stress and maintaining tissue homeostasis during regeneration and remineralization. J Periodontol 2014;85:810-818. KEY WORDS Fibroblasts; gingiva; periodontal ligament; proteome. * Division of Periodontology, College of Dentistry, The Ohio State University, Columbus, OH. † Department of Periodontics, College of Dentistry, University of Illinois at Chicago, Chicago, IL.
O
nce thought to be static cells of connective tissues (CTs), fibroblasts have reemerged as dynamic cells responsible for the health, function, and immunity of a tissue. Human fibroblasts demonstrate selfrenewal capacity and multipotent potential, indicating their ability to perform a range of cellular functions beyond basic homeostasis that includes both repair and regeneration.1,2 Because fibroblast attachment to the extracellular matrix (ECM) helps to maintain cell shape and function in addition to tissue integrity, it has been suggested that fibroblast behavior is essentially controlled by a balance of the synthesis and degradation of the ECM.3,4 Although found in many different tissues, fibroblasts have functional characteristics that are unique to their biologic niche. For example, proliferation, mobility, production of ECM, ability to respond to microbial and viral challenges, metabolism and repair of the ECM, and regenerative capacity will vary depending on the tissue source of the fibroblast.5 Considering the importance of the periodontal ligament (PDL) in the maintenance and regeneration of the periodontium, defining cellular behavior of the principal resident of this tissue, the fibroblast, is essential to understanding regulation of the periodontium. Although human gingival fibroblasts (hGFs) and human PDL fibroblasts (hPDLFs) exhibit numerous similarities, it has been posited that secretory and behavioral differences exist between these cell types. From an embryologic
doi: 10.1902/jop.2013.130161
810
J Periodontol • June 2014
viewpoint, hGFs and hPDLFs do not originate from the same tissues. hPDLFs are derived from the inner layer of the dental follicle and are ectomesenchymal in origin.6 Derivation of hGFs is not completely understood, but it is thought that these cells develop from either the dental follicle or embryonic ridge mucosa and are mesenchymal cells.7 In addition to their embryologic origin, hGFs and hPDLFs have been noted in cell culture to differ in proliferative capacity and glycosaminoglycan content,8 chemotactic response,9 apoptotic propensity,10 protein and collagen synthesis,11 as well as the capacity to form mineralized tissue.12 Gene expression profiles were reported to differ between hGFs and hPDLFs, reflecting intrinsic functional differences of the two cell populations and fundamental differences in their tissue-specific activities.10 Because genes affect protein levels and functional differences in cells are manifest at the protein level, functional differences between hGFs and hPDLFs may derive from differences of their respective proteomes. In dentistry, research on the proteome has focused on oral flora and pathogens, salivary secretions, and protein expression of hard tissues. Proteomic biomarkers in caries pathogen profiles have been found to be useful in predicting caries in a pediatric population.13 In endodontic lesions, proteomics has been used to demonstrate the complexity of microbiota involved in the pathogenesis of the necrotic pulp.14 Proteomic analysis of dental hard tissues has been used to describe the protein expression of dentin to define the organic matrix of dentin and to characterize odontoblasts.15 In a review of published studies regarding proteomes of oral soft tissues, only three articles pertain to the human gingiva or periodontium.16-18 These studies are broad protein surveys of the PDL,16 neutrophils of patients with aggressive periodontitis,17 and/or drug-associated enlargement of gingival tissues.18 Unlike the broad surveys of proteins in tissues or cells, the purpose of this study focuses on membrane-bound and membraneassociated proteins found in either hGFs or hPDLFs. Although membrane-bound proteins are difficult to isolate, their influence on cell function is significant, because these are the proteins through which cells monitor their environments, maintain homeostasis, and signal changes to the nucleus that ultimately affect cellular and biologic responses to the monitored environment.19,20 Membrane-bound and/or -associated proteins not only are necessary for numerous cellular functions, changes or disruptions to them drive signal transduction, substantially affecting tissue function and the health of the individual. For example, disruptions in nuclear lamina,21 an essential element of the nuclear envelope, have been implicated in Emery-Drifuss muscular dystrophy, autosomal
McKnight, Kelsey, Hooper, Hart, Mariotti
recessive Charcot-Marie-Tooth disorder type 2B1, and autosomal recessive manibuloacral dysplasia.22 Considering how important membrane proteins are for cellular function, this investigation was designed to evaluate the putative differences in the membrane-bound and -associated proteome of hGFs and hPDLFs. MATERIALS AND METHODS Collection of Samples Fibroblasts were harvested from human gingiva and hPDLs of individuals with planned extraction of impacted third molars at the Division of Oral and Maxillofacial Surgery at The Ohio State University College of Dentistry. Teeth included for the harvest of PDLFs had three-fourths or more root formation and were completely or partially bony impacted with radiographic evidence of a dental follicle present and lack of communication with the oral cavity. Potential participants had a diagnosis of gingival health, which was confirmed by the absence of gingival inflammation or infection. Interested participants who met inclusion criteria provided signature of informed consent. This study was approved by the Institutional Review Board of The Ohio State University. Inclusion criteria for this study were as follows: 1) healthy patients without a history of systemic disease; 2) aged 16 years or older; 3) a patient of record of The Ohio State University College of Dentistry; and 4) a previously signed treatment plan for extraction of third molar(s). Tissues from four individuals (one male and three females, aged 16 to 36 years; mean age: 20.1 years) who consented to participate were used. Culture of Fibroblasts Four matched pairs of hGFs and hPDLFs were cultured as described previously.23 Briefly, hGFs were harvested from the gingiva of the overlying flap of the impacted third molars. All gingival samples appeared clinically healthy (firm, non-edematous, and coral pink) at biopsy. hPDLFs were harvested from the middle third of the root of impacted third molars. At the time of sampling, explants were immediately placed in 10 mL biopsy media, which consisted of Eagle minimal essential medium supplemented with 10% fetal bovine serum, 350 mg/mL L-glutamine, 100 mg/mL penicillin, and 100 mg/mL streptomycin. Fibroblasts were propagated in 25-cm2 flasks in media at 37C in a humidified incubator with 5% CO2 and 95% air atmosphere. Media were exchanged in the flasks every 48 hours. All experiments were performed only on cells in the fourth passage. Isolation and Solubilization of Membrane-Bound and -Associated Proteins Before confluence, enrichment of membrane-bound and -associated proteins from cells of the fourth 811
Periodontal Cell Proteome
passage was undertaken according to the modified protocol of Chevallet et al.24 Briefly, cell pellets were suspended in 40 mM Tris-HCl (pH 8.0) with protease inhibitor at a concentration of 200 mL/1 mL A600 pellet size and transferred to a 1.5-mL microcentrifuge tube for cell fraction by sonication on ice with medium power for three cycles of 10 seconds. After fractionation, the cell lysate was centrifuged for 15 minutes at 4C at 10,000 rpm to pellet insoluble cellular components. The pellet of the insoluble proteins was resuspended in 200 mL lysis buffer consisting of 40 mM Tris-HCl (pH 8.0), 7 M urea, 2 M thiourea, 0.25% weight-to-volume amidosulfobetaine-14, and 0.25% of 100% Nonidet-40 and incubated at 22C for 30 minutes. Subsequently, samples were mixed for 1 minute and centrifuged (13,200 rpm) for 30 minutes at 22C. The supernatant, containing the membrane proteins with some lipid, nucleic acid, and sugar components, was diluted to 500 mL with distilled water. Twenty percent of fresh trichloroacetic acid was added to suspension and incubated on ice for 1 hour to remove contaminants and centrifuged (13,200 rpm) for 15 minutes. The pellet was recovered, and this procedure was repeated. Without disturbing the pellet, 10 mL supersaturated Tris base was added to the pellet. After 5 minutes of incubation, 1 mL iced acetone was added to the pellet and mixed, and the protein suspension was centrifuged (13,200 rpm) for 15 minutes at 4C. The acetone was removed, and the pellet was allowed to air dry and resuspended in 2 mL/200 mL protein solubilizer‡ and 2 M urea. Peptide Separation, Protein Identification, and Quantitation The processed protein samples were submitted for enzyme digestion and identification at the Mass Spectrometry and Proteomics Facility at The Ohio State University for label-free proteomic quantitation methodology. Briefly, protein was reduced by adding 10 mL dithiothreitol (5 mg/mL solution prepared in 100 mM ammonium bicarbonate), and the reaction proceeded at 60C for 30 minutes. To block reduced cysteine residues, 10 mL iodoacetamide solution (15 mg/mL in 100 mM ammonium bicarbonate) was added to the sample and allowed to incubate at room temperature for 15 minutes in the dark. Trypsin was prepared in 50 mM ammonium bicarbonate and added to the protein solution with an enzyme to substrate ratio of 1:25 (weight-to-volume). The sample was incubated for 2 hours at 37C before being quenched by acidification. Capillary-liquid chromatography-nanospray tandem mass spectrometry (LC-MS/MS) of global protein identification was performed on an orbitrap mass 812
Volume 85 • Number 6
spectrometer§ equipped with a microspray source operating in positive ion mode. Samples were separated on a capillary columnii using a high-performance liquid chromatography system.¶ Each sample was injected into the precolumn cartridge# and desalted with 50 mM acetic acid for 10 minutes. After injection, the peptides were eluted off of the trap onto the column. Mobile phase A was 0.1% formic acid in water, and 0.1% formic acid in acetonitrile was used as mobile phase B. Flow rate was set at 2 mL/minute. Typically, mobile phase B was increased from 2% to 50% in 90 to 250 minutes, depending on the complexity of the sample, to separate the peptides. Mobile phase B was then increased from 50% to 90% in 5 minutes and kept at 90% for another 5 minutes before being brought back quickly to 2% in 1 minute. The column was equilibrated at 2% of mobile phase B (or 98% mobile phase A) for 30 minutes before the next sample injection. MS/MS data were acquired with a spray voltage of 2 kV at a capillary temperature of 175C. The scan sequence of the mass spectrometer was based on the data-dependent TopTen method: the analysis was programmed for a full scan recorded from 300 to 2,000 Da and a MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive scans of the 10 most abundant peaks in the spectrum. The resolution of the full scan was set at 30,000 to achieve high mass accuracy MS determination. The collusion-induced dissociation fragmentation energy was set to 35%. Dynamic exclusion is enabled with a repeat count of 30 seconds, exclusion duration of 350 seconds, a low mass width of 0.50 Da, and high mass width of 1.50 Da. Multiple MS/MS detection of the same peptide was excluded after detecting it three times. Sequence information from the MS/MS data was processed by converting the raw (.raw) files into a merged file (.mgf) using an in-house program.** The resulting .mgf files were searched,†† and the database was searched against protein sequence databases.25,26 The mass accuracy of the precursor ions was set to 2.0 Da given that the data were acquired on an ion trap mass analyzer, and the fragment mass accuracy was set to 0.5 Da. Considered modifications were methionine oxidation and carbamidomethyl cysteine. A proteome software‡‡27 ‡
Invitrosol protein solublizer, Invitrogen, Thermo Fisher Scientific, Waltham, MA. § Finnigan LTQ orbitrap mass spectrometer, Thermo Fisher Scientific. ˚ , Michrom Bioresources, Auburn, ii 0.2 · 150 mm Magic C18AQ 3m 200A CA. ¶ UltiMate 3000 HPLC system, Thermo Fisher Scientific. # Thermo Scientific Scientific. ** RAW2MZXML_n_MGF_batch (merge.pl, a Perl script). Maintained by the Mass Spectrometry and Proteomics Facility at The Ohio State University, Columbus, Ohio. †† Mascot Daemon v.2.3.2, Matrix Science, Boston, MA. ‡‡ Scaffold, Proteome Software, Portland, OR.
J Periodontol • June 2014
was used to validate protein identifications derived from MS/MS sequencing results. The software verifies peptide identifications assigned by mass spectrometry data analysis programs§§iiii and then probabilistically validates these peptide identifications and derives corresponding protein probabilities. Protein Function and Ontology Protein ontology was assessed using literature reports of protein functions as well as Web-based tools28,29 to identify functional annotation to characterize molecular functions and biologic processes that were used in reporting the potential relationships and interconnecting ontologies for proteins determined to show differential expression in hGFs and hPDLFs. Statistical Analyses The mean, standard deviation, confidence interval, paired t test, and Fisher exact test for all proteins were determined using the datasets of the four matched hGF and hPDLF membrane pellets using bioinformatic software and statistical program analysis by the proteome software. Algorithms for calculating protein probabilities from peptide probabilities were performed using the proteome software. RESULTS Four matched pairs of hGF and hPDLF strains were successfully propagated and underwent protein isolation and proteomic analysis from fibroblasts derived from the overlying gingiva of impacted third molars and the PDL of the impacted third molars. The four source individuals ranged in age from 16.4 to 25.4 years, with a mean age of 20.1 years. Three source individuals were female, and one was male. All participants were systemically healthy with no medication use reported, with the exception of one female, who reported taking an oral contraceptive. All participants were never-smokers. Intraoral examinations determined that all participants were free of any signs or symptoms of gingival inflammation, periodontal disease, or oral infections. A total of 518 proteins were identified from the eight samples analyzed via LC-MS/MS (dataset identifier PXD000387 and DOI 10.6.019/PXD000387).30 Four hundred fifty proteins were common to both hGF and hPDLF. Of the proteins identified, 214 were known membrane-bound or -associated proteins (dataset identifier PXD000387 and DOI 10.6.019/ PXD000387),30 and 165 proteins were known nuclearassociated proteins (dataset identifier PXD000387 and DOI 10.6.019/PXD000387).30 Twenty-seven proteins, identified from the 450 proteins common to both hGF and hPDLF, were detected in statistically significantly greater quantities in either hGFs or hPDLFs (Figs. 1 and 2). Thirteen proteins were detected in significantly greater
McKnight, Kelsey, Hooper, Hart, Mariotti
quantities in hGFs (Figs. 1 and 2). These proteins included aminopeptidase N, A-kinase anchor protein 2, annexin A6, annexin A11, UPF0556 protein C19orf10, prohibitin-2, nicotinamide adenine dinucleotide (NADH)-ubiquinone oxidoreductase 75 kDa mitochondrial, protein AHNAK2, annexin A4, acetylcoenzyme A (CoA) acetyltransferase mitochondrial, microtubule-associated protein 4, Src homology 3 (SH3) domain-binding glutamic acid-rich-like protein 3, and trifunctional enzyme subunit b mitochondrial. Four membrane or associated proteins were detected in greater quantities in hGFs compared with hPDLFs (Fig. 1). These proteins include annexin A4, aminopeptidase N, prohibitin-2, and NADH-ubiquinone oxidoreductase 75 kDa subunit. The three proteins that were detected in greater quantities in the hGFs that were nuclear-associated proteins were annexin A11, protein AHNAK2, and SH3 domain-binding glutamic acid-rich-like protein 3 (Fig. 1). Fourteen proteins were detected in significantly greater quantities in hPDLFs compared with hGFs (Figs. 1 and 2). These proteins include a-2-HSglycoprotein, calcium/calmodulin-dependent protein kinase type II subunit d, desmoplakin, serum albumin, membrane-associated progesterone receptor component 2, basement membrane-specific heparin sulfate proteoglycan core protein, epidermal growth factor (EGF)-like repeat and discoidin I-like domaincontaining protein 3, filaggrin, voltage-dependent calcium channel subunit a-2/d-1, 4F2 cell-surface antigen heavy chain, nucleobindin-1, four and a half LIM domains protein 2, ubiquitin carboxyl-terminal hydrolase isozyme L1, and profilin-1. Nine of the proteins identified preferentially in hPDLFs compared with hGFs were membrane-bound or -associated proteins (Fig. 1). These proteins include calcium/ calmodulin-dependent protein kinase type II subunit d, desmoplakin, voltage-dependent calcium channel subunit a-2/d-1, 4F2 cell-surface antigen heavy chain, nucleobindin-1, membrane-associated progesterone receptor component 2, four and a half LIM domains protein 2, and ubiquitin carboxyl-terminal hydrolase isozyme L1. The remaining three proteins that were detected in greater quantities in the hPDLFs that were nuclear-associated proteins were four and half LIM domains protein 2, calcium/calmodulindependent protein kinase type II subunit d, and ubiquitin carboxyl-terminal hydrolase isozyme L1 (Fig. 1). Forty of the 518 proteins identified by proteomic analysis were only detected in hGFs (Table 1). Of the 40 proteins, 18 were membrane-bound or -associated proteins (Table 1). Twenty-nine of the 518 proteins identified by proteomic analysis were only §§ SEQUEST, Thermo Fisher Scientific. iiii Mascot Daemon v.2.3.2, Matrix Science.
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Figure 1.
Membrane/nuclear-associated proteins in the hGFs and hPDLFs. Data are expressed as the mean – SD. All proteins listed exhibit a statistically significant difference (P
