Identification of Protein Components in in vivo Human Acquired

The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva. The structural analysis of this integume...
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Identification of Protein Components in in vivo Human Acquired Enamel Pellicle Using LC-ESI-MS/MS Walter L. Siqueira,† Weimin Zhang,† Eva J. Helmerhorst,† Steven P. Gygi,‡ and Frank G. Oppenheim*,† Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, Massachusetts 02118, and Department of Cell Biology, Harvard Medical School, Harvard University, 240 Longwood Avenue, Boston, Massachusetts 02115 Received November 2, 2006

The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva. The structural analysis of this integument relies on efficient pellicle harvesting and protein identification procedures. Material from three individual subjects and two pooled samples yielded the identification by LC-ESI-MS/MS of 130 pellicle proteins of which 89 were found in three or more experiments. A high intersubject consistency in pellicle composition was observed. Keywords: acquired enamel pellicle • proteomics • oral • mass spectrometry • LC-ESI-MS/MS • linear ion trap • proteins • saliva

Introduction In recent salivary research, proteomics has become a critical tool to gain insights into complex proteomes in oral physiology and pathology and to identify biomarkers for systemic and oral diseases.1-6 An important structure present in the oral cavity is called the acquired enamel pellicle (AEP). This integument is formed by the selective adsorption of proteins, peptides, and other molecules present in oral fluid onto the enamel surface.7 The AEP forms an interface between the tooth surface and the oral environment and acts as a selective permeability barrier that regulates demineralization/remineralization processes. The AEP also dictates the composition of initial microbial tooth colonizers that ultimately form the oral biofilm and mature plaque.8 Functions of AEP include neutralization of acid produced by oral bacteria and acting as a lubrication film, thereby protecting the teeth from abrasive forces.9 The investigation of the composition of AEP composition, being so closely attached to tooth surfaces, is of extreme importance in understanding its role in the dental decay process and for the discovery of biomarkers for dental caries activity or periodontal disease. The identification of the composition of AEP as opposed to that of salivary secretions is more challenging due to the fact that only minute amounts of AEP can be collected from tooth surfaces. These amounts have been estimated to be approximately 0.5-1 µg per tooth surface.10 Therefore, initial attempts to gain insights into this structure have been carried out using hydroxyapatite discs or bovine/human enamel slices * To whom correspondence should be addressed. Frank G. Oppenheim, D.M.D., Ph.D., Boston University Goldman School of Dental Medicine, Dept. Periodontology and Oral Biology, 700 Albany Street, CABR, Suite W-201, Boston, MA 02118. Phone: 617-638-4756. Fax: 617-638-4924. E-mail: [email protected]. † Boston University. ‡ Harvard University.

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mounted on removable devices in the oral cavity. These in vitro and in situ studies have provided valuable information on salivary proteins that display affinity for hydroxyapatite surfaces.11-20 Preliminary insights in in vivo AEP protein composition stem from studies in which the pellicle formed on tooth surfaces was removed by chemical and mechanical means followed by protein identification.21-27 Our group has initiated the characterization of in vivo AEP by immunizing mice and utilizing the amplification of the immune system to obtain detectable reactivities toward the components present in AEP. Using this approach, 18 in vivo AEP proteins were identified.28,29 In subsequent studies employing proteomics technologies, we demonstrated that at least 200 protein spots are detectable in AEP in a 2-D gel electrophoretogram, and we initiated studies for their identification.30 General limitations of 2-D PAGE are the dependence on protein separation, the visualization of proteins in the gel, and the labor-intensive processing of excised protein spots.31 Over the past few years “shotgun proteomics”32 methods have gained increasing interest in the field of proteomics. This method reduces sample handling time and eliminates the need for processing of individual proteins. It allows the direct analysis of extremely complex biological samples and rapidly generates a protein profile and sequence information. This study describes the results of the application of this technology for the analysis of in vivo formed AEP.

Materials and Methods Human Subjects. AEP was obtained from healthy nonsmoking male and female volunteers, ranging in age from 24 to 40 years. The subjects exhibited neither gingivitis, periodontal disease, active dental caries, nor any other oral condition that could affect oral fluid composition. AEP collection proto10.1021/pr060580k CCC: $37.00

 2007 American Chemical Society

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Proteomics of the Human Acquired Enamel Pellicle

cols were approved by the Institutional Review Board of Boston University Medical Center, and informed consent was obtained from each subject participating in the study. AEP Collection. Each donor was subjected to a meticulous dental prophylaxis employing coarse pumice containing no additives (Preppies, Whip Mix, Louisville, KY). AEP was then allowed to form naturally on the enamel surface over a 2 h time period. During this period, the participants were asked to refrain from any consumption of food or beverages, other than water. After 2 h, teeth from each quadrant were isolated with cotton rolls, washed with water, and dried by air. For the collection of AEP material, a dry collection strip of 0.5 cm × 1.0 cm (electrode wick filter paper, Bio-Rad, Hercules, CA, catalog # 1654071) was folded such that half of the surface could be brought in contact with the tooth surface using a dental forceps (Hu-Friedy, Chicago, IL). The strip was used to swipe the coronal two-thirds of the labial/buccal surfaces, spanning from the central incisor to the first molar. In each quadrant, a separate collection strip was used for AEP collection. A total of four collection strips from each participant were placed into a polypropylene microcentrifuge tube and kept frozen at -20 °C until used for electrophoresis. AEP was collected from 3 donors. Materials collected on the first day (total 12 collection strips) were pooled, materials collected on the second day were also pooled, and materials collected from each subject on the third day were kept separate. AEP Extraction by SDS-PAGE. To recover AEP proteins from the collection strip, 30 µL of sample buffer containing 0.125 M Tris-HCl, 4% SDS, 2% glycerol, 10% 2-mercaptoethanol was added to the microcentrifuge tube containing either 4 collection strips that were randomly selected from the pool of 12 strips used to collect AEP material from 3 subjects or 4 collection strips from individual subjects. After boiling for 5 min, the collection strip along with sample buffer were placed directly in one of the 18 wells of a precast 12% SDS-PAGE lane (BioRad, Hercules, CA). Gel electrophoresis was carried out for 15 min, the time necessary for all the AEP proteins to enter the gel. There was no intention to optimize the separation of individual proteins. During electrophoresis, the voltage was kept constant at 120 V. A collection strip containing no protein was used as a control to identify potential non-pellicle derived proteins in our analyses. The efficiency of protein recovery was examined by applying 10 µL of a protein molecular weight (MW) standard (Precision Plus protein standard, Bio-Rad, Hercules, CA) directly on a collection strip and placing the strip onto the gel followed by polyacrylamide gel electrophoresis (PAGE). Amounts recovered were compared to 10 µL of MW standard directly loaded on the gel. Protein amounts in the gel were determined by scanning the gels using a proteomics imaging system (VersaDoc 3000, Bio-Rad, Hercules CA) followed by densitometric analysis of the gels using Quantity One software (Bio-Rad, Hercules, CA). In-Gel AEP Digestion. After staining the gel for 30 min with Coomassie Brilliant Blue R-250 and destaining in methanol/ acetic acid/water (40/10/60% v/v) for 2 h, the stained areas of the developed lanes were each excised with a new razor blade and subsequently cut in two parts. Excised gel slices were each cut into approximately 1 mm3 pieces. Gel pieces were then subjected to a modified in-gel trypsin digestion procedure. The trypsinization was carried out in 50 mM ammonium bicarbonate solution containing 12.5 ng/µL of modified sequencinggrade trypsin (Promega, Madison, WI). Peptide extraction was

achieved by multiple wash and hydratation steps as described by Shevchenko et al., 1996.33 LC-ESI-MS/MS Analysis. Mass spectrometric analysis of the extracted peptides was carried out using a nanoscale reverse-phase HPLC capillary column, which was created by packing 5 µm C18 spherical silica beads into a fused silica capillary with a flame-drawn tip. A gradient was formed using increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid) from 8 to 42% in 21 min at a flow rate of approximately 140 nL/min. The gradient was provided by an Agilent 1100 series binary HPLC pump (Agilent Technologies, Palo Alto, CA). The electrospray voltage was set to 1.8 kV. Mass spectrometric analysis were carried out on a LTQ linear ion trap (Thermo-Finnigan, San Jose, CA), which was operated in the positive-ion mode using data-dependent acquisition methods initiated by a survey MS scan in the range of m/z 4001300, which was followed by a MS/MS analysis of selected peptide ions. Data Analysis. The obtained MS/MS spectra were searched against human protein databases. (Swiss Prot and TrEMBL, Swiss Institute of Bioinformatics, Geneva, Switzerland, http:// expasy.org/sprot) using SEQUEST (Bioworks Browser 3.2, Thermo-Finnigan, San Jose, CA). The searches were performed by selecting the following SEQUEST parameters: (1) No specific protease enzyme, (2) 2 Da precursor ion mass tolerances, (3) 1 Da fragment ion mass tolerance, and (4) variable modifications of oxidized methionine. The SEQUEST score filter criteria applied to the MS/MS spectra were: XCorr score > 1.9 and 2.6 for Z ) 2 and 3, respectively. A dCorr value threshold of 0.1 was applied. Any non-tryptic peptides passing the filter criteria were discarded because it has been shown that the majority of these peptide identifications are incorrect.34 Finally, only proteins for which two or more unique peptides were identified are reported in this study.

Results and Discussion With the new pellicle recovery method, we investigated the possibility to transfer proteins directly from a collection strip into an SDS gel using the force of an electric field. The efficiency of eluting proteins from collection strips into the gel by this method was assessed by comparing individual protein band intensities of 10 µL solution containing 10 standard proteins applied directly to the gel with 10 µL containing the same proteins but applied to a collection strip followed by SDS-PAGE. The results indicated that the average percentage of protein recovered from the collection strip was 97% (Figure 1A, lanes 1 and 2, and Figure 1B). Figure 1A, lane 3 shows AEP proteins recovered from 4 collection strips collected from one subject. The strong staining over the entire MW range further exemplifies an excellent protein elution from the collection strip and the presence of many proteins in pellicle differing only slightly in size. Having established that proteins can be recovered from collection strips at a high efficiency, we randomly selected 4 strips from a total of 12 strips containing pellicle material from 3 subjects and subjected these strips to the same procedure with the exception that gel electrophoresis was carried out for only 15 min. The purpose of using SDS-PAGE was just to facilitate protease digestion. It is believed that proteins in such a denaturing gel matrix will be in an open structure, which increases the tryptic digestion efficiency.35,36 After trypsinization, peptides were extracted from the gel and subjected to nanoscale LC-ESI-MS/MS. The base-peak chromatogram for Journal of Proteome Research • Vol. 6, No. 6, 2007 2153

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Figure 1. Assessment of the efficiency of protein recovery from collection strips. (A) SDS-PAGE and Commassie Brilliant Blue staining of proteins from collection strips. Lane 1, 10 µL of a MW standard (10-250 kD) spotted on collection strips. Lane 2, 10 µL of the MW standard applied directly on the gel. Lane 3, four collection strips containing AEP proteins and sample buffer. Lane 4, collection strip devoid of protein with sample buffer. (B) Optical density measurements of each of the proteins in the MW standard recovered from the collection strip (9) and from solution (0).

Figure 2. Base-peak chromatogram of a pooled AEP sample. Peptides generated by trypsinization were recovered from the gel, loaded on a nanoscale RP-HPLC column, and eluted in a gradient from 8 to 42% buffer B in 21 min. On the y-axis, the peptide ion peak intensity is plotted.

reversed-phase chromatography monitored by the mass spectrometer represents the intensity of all peptide ions in the sample in a single scan (Figure 2). The peptide ions were identified by the SEQUEST search following the criteria as described in the Material and Methods. As an example, Figure 3 shows an MS/MS scan of one precursor ion chosen from the survey scan. The matched b and y ions are indicated in the graph. This peptide ion was later identified as a tryptic peptide from a Calmodulin-like protein 3 (Swiss-Prot accession # P27482). By applying these types of analyses, 89 different proteins were identified in the first AEP pool and 78 proteins were identified in the second AEP pool. In total, 96 different proteins were identified in the two pools, yielding a 74% overlap in proteins between these two samples. It should be pointed out that when one AEP sample was analyzed in triplicate, the overlap in identified proteins in any two experiments was 6270% (data not shown), which is in accordance with studies showing that high complexity protein samples overlap by 6776%.34,37 Therefore, the availability of two pooled AEP samples make it difficult to draw conclusions with regard to potential differences in composition between pellicle pools collected on different days. It is remarkable that 71 proteins were identified in both pools. 2154

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Figure 3. MS/MS analysis of a precursor ion with an m/z value of 943.6. The precursor ion with an m/z value of 943.6 was one of five peptide ions chosen for collision-induced dissociation from a survey scan. It was later identified as a tryptic peptide from Calmodulin-like protein 3. Matching b and y ions are indicated in the graph.

To further investigate the reproducibility of AEP composition, individual pellicle samples from the same donors were subjected to LC-ESI-MS/MS. In these individual analyses, 105, 99, and 89 proteins were identified. These results showed that with the new sample collection technique, sufficient material can be obtained to gain insights into the pellicle composition from a single individual. Comparisons between identified proteins in individual and pooled pellicle samples showed 81, 89, and 84% overlap of proteins for subjects 1, 2, and 3, respectively (Table 1). These data together indicate that the composition of AEP shows a high level of consistency among subjects, which supports our observations in previous studies using an in vitro model for pellicle formation.38 It also supports the contention that the adsorption of AEP proteins to the enamel surface is a specific process21,23 given the high overlap in proteins identified and the much larger numbers of proteins that are present in oral fluid surrounding teeth.1-5

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Proteomics of the Human Acquired Enamel Pellicle Table 1. Summary of Proteomics Analyses of AEP Samples samples

# proteins identified

overlap with pooled sample (%)

pooled samplesa subject 1b subject 2b

96 105 89

81 89

99

84

subject 3b

total identified proteins

}

130

a Pellicle material was collected from 3 subjects, pooled, and analyzed by LC-ESI-MS/MS. The results shown represent the total number of proteins identified in two independent experiments. b Pellicle material from individual subjects were analyzed in separate experiments, one experiment per subject.

The combination of all 5 experiments (pooled and individual analyses) yielded a total of 130 different pellicle proteins/ peptides that were identified by at least two peptides. Table 2 shows all of the identified proteins, which are listed and ordered by the number of peptides identified and reporting their primary accession number of the Swiss-Prot database. The last column of Table 2 illustrates the number of times that each protein was identified in the 5 independent experiments. The majority of the proteins (89 in total) were identified in at least 3 experiments, indicating a high overlap in AEP protein composition of the 5 AEP samples analyzed in this study. Among the identified 130 proteins, 17 proteins had been reported in previous in vivo studies on AEP.9,21,28-30 These 17 proteins are listed in Table 2 in bold. Overall, this study has led to the discovery of 113 new pellicle proteins contributing to the formation of AEP. Notably, there were also some proteins that had been identified in in vivo AEP studies before but were not found in this study using the in-gel digestion approach. These proteins comprise members of the histatin, statherin, and acidic prolinerich protein (PRP) family. Histatins, statherins, and acidic PRPs have been shown to be among the first proteins that adsorb onto hydroxyapatite in a number of in vitro studies.39-41 The presence of these proteins in in vivo AEP was confirmed by our group using immunochemical approaches.28,29 With the current MS analysis of tryptic digests, none of the polymorphic species of these proteins were found, not even when all generated peptides were manually screened for possible tryptic and non-tryptic peptides. Therefore, the sample processing method was adjusted, and proteins attached to the collection strip were trypsinized directly using a solution containing 12.5 ng/µL trypsin in 50 mM ammonium bicarbonate buffer. After digestion of the samples for 16 h at 37 °C under nonreducing conditions, the generated peptides were extracted from the strip in water, and LC-ESI-MS/MS analysis and data processing were carried out as described before. For partial tryptic peptides, only double and triple charge peptides were considered and the Xcorr value was set at 3.0 for Z ) 2 and at 4.0 for Z ) 3 to meet the goal of a false positive rate of less than 1%.42 This latter approach of direct trypsinization led to the identification of five different PRP peptides present in multiple isoforms of acidic PRPs. It has been reported that proteolytic cleavage of these proline-rich proteins does not always occurs at the tryptic site,43 and our results confirmed this observation. Only one tryptic peptide (K.PQGPPQQGGHPPPPQGR.P, P91R107) was observed. The other four peptides were partial tryptic peptides (tryptic cleavage either at N or C terminus) corresponding to G93-R107, P108-P121, P91-Q109, and G35-R46 in the acidic PRP family (Swiss-Prot accession # P02810). All of these

peptides were doubly charged. The tandem mass spectrum of the partial tryptic peptide G93-R107 is illustrated in Figure 4. In this spectrum the y5, y6, y132+, and y142+ ions are more abundant than the other b and y ions, which clearly illustrated the reported proline effect which is due to the fact that the N-terminal bond of proline is more sensitive to CID than the C-terminal bond.44 Also, the yield of y ions was almost complete whereas only half of the theoretical b ions were observed. The results of this experiment show that PRPs are indeed part of the AEP proteome but are not readily detectable following ingel digestion. In contrast to acidic PRPs, histatins and statherins were neither detected in samples digested with trypsin in the gel or in samples digested with trypsin directly on the collection strip. A possible explanation for this observation is that the high abundance of arginine and lysine residues in the N-terminal region of these proteins will generate very small sized tryptic peptides with m/z values below 400 that were not detected. Keratins are important proteins in the oral cavity because both gingival and oral mucosa are rich sources for these types of proteins. It is therefore not surprising that keratins have been reported in the acquired enamel pellicle.30 In this study, we identified 18 different keratins. Eleven of these keratins have been reported to be present in saliva, in vivo AEP, serum, and oral epithelia3,30,45 and may therefore truly participate in the formation of AEP. Keratins are normally found in skin and hair and are detected frequently as contaminants due to the high sensitivity of the MS technology. In a control experiment using a blank collection strip, five keratins could be identified by us that have not been reported in oral sources. Therefore, these five keratins were considered possible contaminants and were excluded from Table 2. Overall, our data indicate the presence of 13 keratin proteins that may represent legitimate constituents of AEP. A long held tenet in pellicle research is that the structure of AEP is derived by an adsorption process of salivary proteins. When the proteins identified in this study were analyzed with respect to their origins, a surprisingly low value of 14.4% of all proteins derived from exocrine salivary secretions. Most of the identified AEP proteins originated from the non-exocrine contributors to whole saliva, comprising cells (67.8%) and serum (17.8%) (Figure 5A). The latter oral fluid contributor enters the oral cavity through the gingival crevicular crevice. In the absence of quantitative data, it is difficult to conclude, but still likely, that the bulk of the pellicle structure originates from salivary proteins, although non-salivary proteins significantly outnumber the exocrine constituents. In addition, it is feasible that more proteins will be identified in salivary glandular secretions in the future and that some of the AEP proteins that are assigned in this study as being derived from cells or serum are actually true salivary secretory proteins. When the identified proteins are grouped based on their possible role in AEP structure formation, AEP proteins can be separated into three main groups (Figure 5B). The first group consists of proteins that have the ability to bind calcium ions, comprising 17.5% of the identified AEP proteins. Among these are the S100 calcium binding protein family and members of the annexin family. These proteins might specifically interact with calcium ions on the enamel surface and can therefore be considered the pellicle precursor proteins. The second group (15.4%) consists of proteins that show a high tendency to bind phosphate ions. Examples of proteins in this group are elongation factor 2 and myosin-9. The phosphate and calcium binding proteins may form the primary protein layer, which adsorbs Journal of Proteome Research • Vol. 6, No. 6, 2007 2155

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Table 2. Proteins Identified in in vivo Acquired Enamel Pellicle Using LC-ESI-MS/MS

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primary accession numbera

peptides identified

maximum coverageb

protein namec

number of times identifiedd

Q01546 P02538 P04745 P04083 P13646 P02768 P02533 P05164 P06702 P08779 P12035 P02788 P15924 Q6uw28 Q9HC84 P07355 P19013 q04695 Q08188 Q8n613 P01857 P04792 P06733 P07737 P61626 Q06830 Q13707 Q53g76 P01876 P02647 P09211 P31151 P31947 P62807 P01009 P04259 P04406 P08311 P13796 P18669 P27482 P31946 P52209 Q561v9 Q6fgb8 Q9hcy8 P30044 P06744 P02787 P00338 P01036 P01040 P01834 P01842 P04080 P05109 P12429 P13639 P20160 P23528 P24158 P28001 P29508 P31949 P35579 P47929 P63104 Q05639 Q99456 Q9ubc9 Q96DR5 Q6ZW52 Q04695 P60174 P36952 P29401 P25311 P23280 P17213 P02675 P01024

20 18 16 14 14 13 12 12 11 11 11 10 9 9 9 8 8 8 8 8 7 7 6 6 6 6 6 6 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

46.0 41.0 42.4 47.5 28.0 23.2 27.0 20.0 87.7 20.0 37.0 15.1 6.6 61.8 2.0 28.1 16.0 26.0 19.5 32.7 31.5 48.3 24.2 56.1 29.1 33.2 18.8 27.5 16.4 22.8 34.4 53.0 27.0 27.2 14.1 12.0 20.1 11.8 11.8 23.3 37.8 15.1 13.3 21.9 24.3 42.3 27.2 14.5 7.7 13.9 29.8 37.8 49.1 32.4 45.5 64.5 12.4 5.8 19.5 27.3 17.6 27.1 9.0 23.8 2.8 30.4 14.7 6.7 8.0 18.9 10.5 7.1 11.0 16.7 12.8 7.3 13.7 10.7 9.4 10.8 3.0

keratin, type II cytoskeletal 2 oral keratin, type II cytoskeletal 6A alpha-amylase A1(salivary)18,20,22,27-29,54 annexin a1 (annexin i) keratin, type I cytoskeletal 1330 serum albumin10,19,29,54 keratin, type I cytoskeletal 14 myeloperoxidase precursor calgranulin b(S100 calcium-binding protein A9)30 keratin, type I cytoskeletal 16 keratin, type II cytoskeletal 3 lactotransferrin precursor26,28 Desmoplakin hypothetical protein LOC124220 mucin-5B precursor29 annexin A2 keratin, type II cytoskeletal 4 keratin, type I cytoskeletal 17 protein-glutamine gamma-glutamyltransferase hypothetical protein CRMN IGHG1 protein ig gamma-1 chain c region heat-shock protein beta-1 alpha-enolase profilin-1 lysozyme c22,26-29 peroxiredoxin-1 acta2 protein beta actin variant Ig alpha-1 chain c region28 Apolipoprotein a-i precursor glutathione s-transferase p S100 calcium-binding protein A7 protein sigma (stratifin) histone h2b.a/g/h/k/l alpha-1-antitrypsin precursor keratin, type II cytoskeletal 6B glyceraldehyde-3-phosphate dehydrogenase cathepsin g plastin-2 phosphoglycerate mutase 1 calmodulin-like protein 3 protein kinase C inhibitor protein 1 6-phosphogluconate dehydrogenase hypothetical protein histone h4 s100 calcium-binding protein a14 (s114) peroxiredoxin-5, mitochondrial precursor glucose-6-phosphate isomerase Serotransferrin precursor l-lactate dehydrogenase a chain cystatin s precursor (salivary acidic protein 1) cystatin a (stefin a) Ig kappa chain c28 Ig lambda chain c regions, IGLC2 protein28 cystatin B calgranulin a S100 calcium-binding protein A8 annexin a3 (placental anticoagulant protein iii) elongation factor 2 azurocidin precursor cofilin-1 (cofilin, non-muscle isoform) myeloblastin precursor histone h2a type 1 squamous cell carcinoma antigen calgizzarin (s100 calcium-binding protein a11) myosin-9 galectin-7 protein kinase C inhibitor protein 1 elongation factor 1-alpha 2 (ef-1-alpha-2) keratin, type I cytoskeletal 12 small proline-rich protein 3 parotid secretory protein CDNA FLJ41598 fis, clone CTONG2025496 heratin, type I cytoskeletal 17 triosephosphate isomerase serpin B5 precursor (Maspin) transketolase zinc-alpha-2-glycoprotein precursor carbonic anhydrase 6 precursor55 bactericidal permeability-increasing protein fibrinogen beta chain precursor18,27,54 complement C3 precursor26

5 5 5 5 5 5 5 5 5 5 5 5 1 5 1 5 5 4 5 5 5 5 4 5 5 4 5 4 5 5 5 4 5 5 4 5 5 5 5 5 4 5 5 4 5 5 3 1 2 5 4 5 5 5 5 5 3 1 5 5 5 5 5 2 5 5 5 4 5 5 5 1 4 2 1 1 1 1 1 2 1

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Proteomics of the Human Acquired Enamel Pellicle Table 2 (Continued) primary accession numbera

peptides identified

maximum coverageb

protein namec

number of times identifiedd

P01037 P01620 P01833 P05783 P06703 P08107 P08727 P08729 P08758 P10599 P12273 P12814 P14618 P19105 P30740 P35754 P61026 P68366 P68371 P80188 P80511 Q16577 Q562l5 Q5hym7 Q6b823 Q6zn66 Q7rtt2 Q8n4f0 Q93081 Q9um07 Q9H4B7 Q3MIH3 Q05524 Q01469 P37837 P37802 P34931 P28676 P28325 P18085 P16401 P11021 P08246 P02774 P02511 P01859 P01591 O75828 O60218

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

24.8 31.2 5.1 4.0 16.7 4.8 7.0 5.0 9.7 23.1 45.9 2.5 6.6 17.1 11.6 38.1 11.0 11.8 9.0 13.1 22.0 11.3 46.6 5.6 41.9 7.0 4.4 9.8 17.0 5.0 4.5 19.3 6.8 12.0 6.6 14.8 4.0 8.5 29.4 16.7 10.2 4.0 6.6 6.8 12.2 10.3 13.9 10.9 8.7

salivary cystatin SA-110 Ig kappa chain v-iii region28 polymeric-immunoglobulin receptor precursor keratin, type I cytoskeletal 18 S100 calcium-binding protein A6 heat shock 70 kD protein 1 keratin, type I cytoskeletal 19 keratin, type II cytoskeletal 7 annexin a5 Thioredoxin prolactin-inducible protein precursor alpha-actinin-1 pyruvate kinase isozymes m1/m2 myosin regulatory light chain 2, leukocyte elastase inhibitor glutaredoxin-1 ras-related protein Rab-10 tubulin alpha-1 chain tubulin beta-2c chain (tubulin beta-2 chain) neutrophil gelatinase-associated lipocalin precursor calgranulin C eukaryotic translation elongation factor 1 actin-like protein hypothetical protein DKFZp686B15196 histone2 h4 hypothetical protein guanylate binding protein family keratin 5b bactericidal/permeability-increasing protein-like 1 histone h3/b protein-arginine deiminase type-4 tubulin beta-1 chain ubiquitin and ribosomal protein L40 alpha-enolase fatty acid-binding protein, epidermal transaldolase transgelin-2 heat shock 70 kDa protein 1L grancalcin cystatin D precursor ADP-ribosylation factor 4 histone H1.5 endoplasmic reticulum lumenal Ca binding protein leukocyte elastase precursor vitamin D-binding protein precursor heat-shock protein beta-5 Ig gamma-2 chain C region18,27,28,54 immunoglobulin J chain28 carbonyl reductase aldo-keto reductase family 1 member B10

4 5 5 3 2 3 4 3 4 4 5 4 5 4 5 1 3 5 5 5 3 1 1 1 5 1 1 4 5 2 3 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 2

a Swiss-Prot accession number. b Percent of the entire protein sequence covered by the various peptides identified. c The identified proteins are listed in order of decreasing numbers of identified peptides. Protein names in bold have been previously identified in in vivo AEP. d In a total of 5 independent experiments.

to the enamel surface because enamel is composed of calcium phosphate salts (hydroxyapatite). The third group (28.2%) consists of proteins that have been described to show interactions with other proteins. Proteins in this group are possibly involved in the formation of the successive protein layers by interacting with proteins directly adsorbed onto the enamel surface. An example of a protein in this group is MUC5B, which has been described to form complexes with several other salivary proteins.46 One of the binding partners of MUC5B is alpha-amylase, which was also identified in this study to be part of AEP. AEP proteins can also be grouped according to putative biological functions such as inflammatory response, antimicrobial, immune defense, buffer capacity, lubrication, and (re)mineralization capacities.7,9 The pie chart in Figure 5C summarizes the result of this categorization. Not surprisingly, there

is a partial overlap in proteins that are involved in remineralization processes and those that have a predicted high affinity for the enamel surface. These proteins include, for example, members of the calgranulin and annexin families. Due to the presence of the calcium binding domain, it is possible that these proteins are directly involved in biologically important remineralization processes affecting the maintenance of enamel integrity. Other proteins identified in AEP are involved in the innate or acquired immune response (11.3%) or exhibit antimicrobial activity (8.3%). Both functions are essential in oral host defense against pathogens. Proteins in this group comprise cystatins (S, SA, A, B, D), lysozyme, lactotransferrin, myeloperoxidase, calgranulin A, and calgranulin B. It is interesting to note that the activities of at least some of these proteins are retained upon adsorption to calcium phosphate minerals.47,48 Journal of Proteome Research • Vol. 6, No. 6, 2007 2157

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Figure 4. MS/MS spectrum of partial tryptic peptide Q.GPPQQGGHPPPPQGR. P from acid salivary proline rich protein 1. Matching b and y ions are indicated in the graph. 1 indicate the possible internal fragments of this peptide.

Figure 5. Classification of AEP proteins by their origins (A), chemical properties (B), and biological functions (C) relevant to AEP structure and function. Sorting of the functions of these proteins was based on their annotations in the database. Proteins from multiple sources were counted multiple times.

Albumin, alpha-1-antitrypsin, cathepsin G, complement C3, and myeloperoxidase have been reported to be strong potential biomarkers for periodontal disease.49,50 The salivary and gingival crevicular fluid concentrations of these proteins either increase or diminish according to the level of periodontal disease.51,52 Because the above-mentioned proteins are part of AEP, the level of inflammatory response related to periodontal disease is likely to have a direct effect on the composition of the pellicle structure. Therefore, this group of proteins, constituting about 13% of the total number of AEP proteins, potentially represents important biomarkers for oral inflammatory diseases.

Conclusions In order to elucidate the proteins that contribute to the composition of in vivo AEP, we identified proteins/peptides using a “shotgun” proteomics approach. A total of 130 proteins were identified to be associated with AEP with high confidence. Of these, 89 proteins were identified in 3 or more of the 5 experiments. The spectrum of components identified was demonstrated to be very consistent because 81, 89, and 84% of proteins in three individual pellicle samples were overlapping with the two pooled samples. The yield and efficiency of pellicle harvesting obtained by the novel collection method shows a protein recovery amenable for analysis of AEP material from a single subject. In future studies, it should be feasible to assess 2158

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pellicles from different regions in the dentition of a single subject. This study provides a comprehensive investigation of proteins that were identifiable in pellicles after trypsinization using the so-called “bottom up” approach. It should be pointed out that a substantial number of proteins in oral fluid are susceptible to proteolytic processing by a variety of proteases that are naturally present in oral fluid.53 It is therefore likely that some of the proteins identified in this study may not be present in the full length polypeptide form within the pellicle structure. Future pellicle studies will have to take this oral fluid proteolysis into account using “top down” approaches by omitting cleavage procedures prior to MS analysis to identify the N- and C-terminal residues of such peptides and to identify other small proteins such as histatins and statherins adhering to the tooth surface. Abbreviations: AEP, acquired enamel pellicle; 2-D, twodimensional; CID, collision-induced dissociation; ESI, electrospray ionization; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/ MS, tandem mass spectrometry; MW, molecular weight; PAGE, polyacrylamide gel electrophoresis; RP, reversed-phase; SDS, sodium dodecyl sulfate.

Acknowledgment. We gratefully acknowledge Dr. Ross Tomaino at the Taplin Biological Mass Spectrometry Facility at Harvard Medical School for help with the LC-ESI-MS/MS

Proteomics of the Human Acquired Enamel Pellicle

experiments and Dr. Erdjan Salih for his advice and help in the preparation of this manuscript. This study was supported by NIH/NIDCR Grants DE05672, DE07652, and DE14950.

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