Mass Spectrometric Analyses of Peptides and Proteins in Human Gingival Crevicular Fluid Luan H. Ngo, Paul D. Veith, Yu-Yen Chen, Dina Chen, Ivan B. Darby, and Eric C. Reynolds* Cooperative Research Centre for Oral Health Science, Bio21 Molecular Science and Biotechnology Institute, Melbourne Dental School, The University of Melbourne, Australia Received August 31, 2009
Gingival crevicular fluid (GCF) is a pathophysiological fluid that flows into the oral cavity. Human GCF was collected using sterile glass microcapillary tubes from inflamed periodontal sites in patients who had a history of periodontal disease and were in the maintenance phase of treatment. Samples from individual sites were analyzed using MS techniques both before and following HPLC. GCF samples were also pooled and subjected to SDS-PAGE, in-gel digestion and MS analyses using both MALDITOF/TOF MS and nanoLC-ESI-MS/MS. MS spectra were used to search human protein sequence databases for protein identification. With these approaches, 33 peptides and 66 proteins were positively identified in human GCF. All of the peptides discovered in this study are reported in GCF here for the first time. Forty-three of the identified proteins, such as actin and the actin binding proteins profilin, cofilin and gelsolin, have not been reported in GCF before. Keywords: gingival crevicular fluid proteome • liquid chromatography mass spectrometry • matrixassisted laser desorption-ionization mass spectrometry • MS/MS tandem mass spectrometry • periodontal disease
Introduction Periodontal diseases comprise a group of diseases affecting the supporting structures of the teeth. These structures include the tissues of the gingiva, the alveolar bone, and tissues of the periodontal ligament. Periodontal diseases are associated with specific bacteria found within the oral cavity as part of the normal flora.1 They are generally characterized by inflammation, destruction of alveolar bone and periodontal ligament, and eventually lead to tooth mobility and tooth loss. Gingival inflammation (a hallmark of gingivitis and often periodontitis) is associated with an increase in the flow of gingival crevicular fluid (GCF) into the periodontal pocket (a physiological space between the coronal gum margin and the tooth, opening into the oral cavity at one end and tapering down to the attachment apparatus). Collection of GCF is a simple and noninvasive procedure. The periodontium is unique in the body in that it provides an interface between a bodily structure (a tooth) which pierces the protection of the epithelium into a bacteria-rich moist environment. Its role is to protect the supporting structures of the teeth from the bacteria in the oral cavity. As such, the periodontium has been shown to have an inflammatory (neutrophilic) infiltrate even in situations of clinical health (i.e., the gingiva appearing healthy).2 Inflammation is associated with an increase in the infiltrate, and if this inflammation becomes chronic, a change in proportion of inflammatory cells, * To whom correspondence should be addressed: Professor Eric C. Reynolds, Cooperative Research Centre for Oral Health Science, Melbourne Dental School, University of Melbourne, 720 Swanston Street, Victoria, 3010, Australia. E-mail:
[email protected]. Fax: +613-9341-1596. 10.1021/pr900775s
2010 American Chemical Society
from a neutrophil dominated infiltrate, to a predominantly lymphocytic lesion occurs. Correlation has been shown between the level of GCF flow and clinical gingival inflammation,3,4 inflammation and pocket depth,5 and clinical gingival inflammation and histological inflammation.6 Histopathological studies on both human2 and animal4,7 tissues have shown that healthy sites do not produce any measurable GCF exudate. GCF contains proteins of serum origin,8 as well as proteins synthesized and secreted in the inflamed periodontal tissues9-11 and other proteins of bacterial origin.9 The site specific nature of GCF (as opposed to saliva) means that it has great potential in containing factors which are specific for the periodontal diseases and may therefore be of diagnostic value. The major protein constituents of GCF have been shown to be albumin, transferrin and IgG.12 Using SDS-PAGE, nonplasma derived proteins were also found in GCF, with molecular weights of 37, 47, 57, and 59 kDa.13 Kojima et al.14 examined GCF (from inflamed gingiva) using 2-D-gel electrophoresis and MS, identifying two nonplasma derived proteins in the lower mass range and found them to be calgranulin A (10.8 kDa) and calgranulin B (13.2 kDa). Calgranulin A has also been identified in GCF using N-terminal amino-acid sequencing of HPLC purified protein.15 More recently, HPLC-ESI-MS has been used for studying peptides and proteins in GCF.16 With this technique, 11 peaks were seen with GCF, and ESI-MS of those peaks resulted in the identification of 8 peptides/proteins based on Mr. These included four alpha-defensins, statherin, PB peptide, cystatin A and albumin. A number of studies have investigated proteins in GCF as possible markers for periodontal disease progression. Using immunoassays, acute phase proteins,17 alkaline phosphatase,18 Journal of Proteome Research 2010, 9, 1683–1693 1683 Published on Web 12/19/2009
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Ngo et al.
Table 1. Clinical Parameters of Sampled Sites sample set
subjects
sites
vol (µL)
30 6 4 6 2
n/a 6 5 8 2
1 2 3
Averaged spectrum (Figure 1) Nano-LC/MS pool (Table 2) Gel - pool 1 (Figure 3a)
9 1 1
4
Gel - pool 2 (Figure 3b)
1
a
PDa (mm)
MGIa
PIa
60.6 (range 43-79) 8 female/1 male 50 F 50 F
4.8 3-5 3-5
3 2-3 2-3
Median 2 (range 0-3) 2-3 2-3
67
3-4
2-3
2-3
age (years)
M
PD ) pocket depth; MGI ) modified gingival index; PI ) plaque index.
dipeptidyl peptidase, bacterial proteases, cathepsin B,19,20 stromelysin-1, tissue inhibitor of metalloproteinase-1,21 collagenase-2,22 gelatinase,23 and collagen breakdown products24-26 (to name a few), have been found in GCF from inflamed sites. A combination of several factors (prostaglandin E2, collagenase, alkaline phosphatase, R-2 macroglobulin, osteocalcin and antigenic elastase) in GCF was shown in a small longitudinal study to have significant diagnostic value (sensitivity, 80%; specificity, 91%) for disease progression as defined by periodontal attachment loss.27 Periodontal disease progression occurs at inflamed periodontal sites. Inflamed sites may have gingivitis or periodontitis. Gingivitis is a reversible inflammatory condition, and does not result in the permanent destruction of the periodontal ligament or alveolar bone. However, in patients with a history of periodontal disease, there is currently no way at a single visit to distinguish between an inflamed site with gingivitis or periodontitis. Periodontitis can at this stage only be diagnosed retrospectively, after the attachment loss has occurred. Clinically healthy sites, as evidenced by the repeated lack of bleeding on gentle periodontal probing at consecutive dental visits, are unlikely to suffer clinical attachment loss.28 From this perspective, it is more relevant to investigate inflamed periodontal sites for their protein composition (and also pristine gingiva produces little or no GCF). A stable site is not necessarily a noninflamed site, as disease progression is not dependent upon pocket depth or level of inflammation per se.29,30 Diagnostic tests are not required to differentiate between a healthy site and an inflamed site as clinical indices of inflammation correspond well with histological inflammation of the periodontium.31 More importantly from a diagnostic point of view is the ability to differentiate between an inflamed but stable site and an inflamed-progressive (active) site. Our aim is to identify peptides and proteins found in human GCF from inflamed sites. Little work has been done in this area to date. Understanding the proteome of GCF from inflamed sites will enhance our understanding of the fate of serum proteins in GCF. In this paper, we report the identification of 33 peptides and 66 proteins in GCF samples using mass spectrometric techniques.
Experimental Section Chemicals. All chemicals used, unless otherwise stated, were purchased from Sigma-Aldrich (St. Louis, MO). Water used was Milli-Q grade (Billerica, MA). Acetone was purchased from EMD Chemicals (Darmstadt, Germany), acetonitrile (ACN) from Riedel-de Hae¨n (Seelze, Germany), and sequencing grade trifluoroacetic acid (TFA) from Agilent Technologies (Palo Alto, CA). GCF Samples. For proteomic analysis, GCF was collected from inflamed sites32 from a total of 12 subjects (Table 1). Subjects were all over 35 years of age, had at least 20 teeth, 1684
sex
Journal of Proteome Research • Vol. 9, No. 4, 2010
had been diagnosed with chronic periodontitis in the past (as per the criteria of the American Association of Periodontology guidelines81) and received treatment for it, and were currently in the maintenance phase of treatment. Informed written consent was obtained from each patient. Exclusion criteria included the use of antibiotics in the past 3 months, pregnancy or lactation, systemic conditions which may affect the progression of periodontitis, any condition requiring premedication prior to treatment, and long-term therapy with nonsteroidal anti-inflammatory drugs. Prior ethics approval was obtained from the Melbourne Research and Innovation office, and the Royal Dental Hospital of Melbourne. GCF was collected from up to five sites per subject (based primarily upon previous pocket depths), with molars (excepting their mesial surfaces) excluded. GCF was collected by trained clinicians using sterile 2 µL glass microcapillary tubes (Drummond Scientific, Brookmall, PA) following the method described by Giannobile et al.33 The use of glass microcapillary tubes was safe for the patient and the operator and was preferred to the use of paperpoints which results in substantial dilution and poor recoveries.34 The site where GCF was to be collected was first isolated if necessary by cotton rolls and gently air-dried to remove any saliva present. Any supragingival plaque was removed with a sterile curet. A sterile glass capillary tube was then placed at the entrance to the periodontal pocket and left for 30 s. Crevicular fluid (0.2-1.5 µL) within the pocket was drawn into the glass tube through capillary action. Samples were placed into microcentrifuge tubes and placed on ice, until final storage at -70 °C. Clinical parameters of the site, including modified gingival indices,32 plaque index,35 and pocket depth and attachment levels, were measured with an electronic constant force probe (Florida Probe) by a single examiner. GCF was dispensed from the microcapillary tube by means of gentle air pressure from one end of the tube, via a bulb. Milli-Q water (1-2 µL) was then drawn into each tube and dispensed with the rest of the sample. MALDI-TOF/TOF Analysis of GCF before and after RP-HPLC Fractionation. Unprocessed GCF samples from inflamed sites were analyzed on an Ultraflex MALDI-TOF/TOF instrument with LIFT II upgrade (Bruker Daltonics, Bremen, Germany) to visualize small proteins and peptides. GCF samples from individual sites were initially analyzed separately. ZipTips (Millipore, Billerica, MA) were used for desalting of samples before direct elution with 50% ACN onto 600 µm anchors on the AnchorChip (Bruker Daltonics) target plate. After spotting of sample, 0.5 µL of matrix (10 mg R-cyano-4hydroxycinnamic acid (CHCA)/1 mL of 50% ACN and 0.1% aqueous TFA) was added and the spot was left to dry before MS analysis. Samples were analyzed in both reflectron and linear modes. Selected intense peaks were analyzed using the MALDI MS/MS function (LIFT) of the mass spectrometer.
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Proteins and Peptides in Human GCF Calibration was performed with a Peptide Calibration mix (#206195) and Protein Calibration Standard I (#206355) for MALDI-MS from Bruker Daltonics (Bremen, Germany). To reveal possible disulfide linkages in proteins/peptides, some GCF samples were prepared in reduced conditions with the addition of dithiothreitol (DTT). After salt removal with ZipTips, DTT was added to the GCF to achieve a final DTT concentration of 10 mM. Samples were heated at 100 °C for 5 min, cooled to room temperature, and spun down at 14 000g for 5 min. These samples were directly spotted onto the AnchorChip target plate and 0.5 µL of CHCA matrix added. Linear mode was chosen for the analysis of these reducible peptides (as spectra of these peptides in reflectron mode were generally poorer), with calibration of the peaks based upon reflectron mode measurements. Alternatively, GCF pooled from 6 sites (6 µL in total) was diluted to 20 µL with 0.1% TFA and centrifuged at 14 000g for 10 min at 4 °C. The supernatant was removed and subjected to RP-HPLC analysis on an UltiMate NanoLC (Dionex LC Packings, Sunnyvale, CA) instrument. The GCF supernatant was concentrated onto a 300 µm i.d. × 1 mm PepMap Nano Precolumn (Dionex LC Packings) before being injected onto a 75 µm i.d. × 15 cm C18 PepMap NanoLC column. The mobile phase consisted of (A) 0.1% aqueous formic acid and (B) 0.1% aqueous formic acid and 80% ACN. Peptides were fractionated using a gradient of 0-100% Buffer B in 40 min at a flow rate of 340 nL/min. Collected A214nm peak fractions were analyzed on the Ultraflex MALDI-TOF/TOF mass spectrometer (as described above) using a dried droplet preparation on an AnchorChip target plate. Each fraction (0.5 µL) was directly spotted onto the AnchorChip target with 0.8 µL of the CHCA matrix solution and allowed to dry. Before analysis, the dried droplet was recrystalized by adding 1 µL of 50% ACN and 0.1% aqueous TFA. Gel Electrophoresis and In-Gel Digestion. For gel electrophoretic analysis, GCF samples (from a single patient) were pooled and clarified by centrifugation at 14 000g at 4 °C. SDSPAGE separation of the proteins was carried out using standard procedures with a Novex 16% polyacrylamide Tricine minigel (Invitrogen, Carlsbad, CA) on an Xcell Surelock Electrophoresis Cell (Invitrogen). A protein marker (#161-0304) SDS-PAGE standard from BioRad Laboratories (Hercules, CA) and BenchMark protein ladder for SDS-PAGE from Invitrogen (Carlsbad, CA) were applied in adjacent lanes to the GCF samples. The conditions used were 126 V (constant) for 90 min. Following staining with Coomassie Blue G-250 overnight, gel bands were cut and placed into labeled microcentrifuge tubes in a laminar flow workbench. In-gel tryptic digestion was performed on each gel band following a method described by Mortz et al.36 Gel pieces were first washed with 900 µL of 50 mM NH4HCO3/ethanol (1:1) for 60 min before dehydration in 1 mL of ethanol for 10 min. Proteins were then reduced in 60 µL of 10 mM DTT/50 mM NH4HCO3 for 60 min at 56 °C. Cysteine carbamidomethylation was performed by adding 60 µL of 55 mM iodoacetamide/50 mM NH4HCO3 for 30 min at room temperature. Gel pieces were washed again and dehydrated. A 10 µL aliquot of modified trypsin (Promega, Mannheim, Germany) (10 ng/µL in 20 mM NH4HCO3 and 1 mM CaCl2) was added for 30 min. Samples were centrifuged and the excess trypsin solution was removed. A 10 µL aliquot of 20 mM NH4HCO3 was finally added and the gel pieces were incubated overnight at 37 °C. Following this, tubes were
centrifuged briefly, and digest solution at the bottom of the tube was removed for analysis. Mass Spectrometric Analyses of In-Gel Digests. In-gel digests (0.5 µL) were acidified with 1% TFA (2.5 µL) and spotted onto a 600 µm AnchorChip target using the CHCA thin layer technique (AnchorChip manual, Bruker Daltonics), and washed with 10 µL of cold 0.1% TFA before being analyzed in the Ultraflex MALDI-TOF/TOF instrument described above. Digest samples (10 µL) were also analyzed on an UltiMate NanoLC system as described in section 2.3 above, but coupled with a nanoelectrospray interface and ion-trap mass spectrometer (esquireHCT; Bruker Daltonics, Bremen, Germany). The instrument was calibrated with ES Tuning Mix (Agilent, Santa Clara, CA). MS analysis of the separated peptides was performed in the MS mode over 300-1500 m/z range and datadependent MS/MS mode of 100-3000 m/z range. MS/MS fragmentation was performed on four of the most intense ions with an active exclusion time of 2 min. Protein Identification. Following MALDI-MS analysis, peaks were detected using flexAnalysis software (Version 2.4, Build 11, Bruker Daltonics, Bremen, Germany). Centroid and SNAP algorithms were used for peak detection for PMF analysis with the following parameters: S/N, 2.5; Maximum number of peaks, 200; Quality factor threshold, 30. DataAnalysis software (Version 3.2, Build 121, Bruker Daltonics, Bremen, Germany) was used for peak detection of LC-MS spectra, with the following parameters: Apex peak detection algorithm; peak width (m/z) 0.1; S/N > 1; relative intensity threshold 0.1%; and an absolute intensity threshold of 100. Using the BioTools software (Version 3.0, Build 1.68, Bruker Daltonics, Bremen, Germany), MS peak lists (ESI-MS/MS, MALDI-MS for PMF, and MALDI-MS/MS) were searched using an in-house Mascot search engine (version 2.1.0) against an NCBI protein sequence database (updated September 20, 2005). Searches were done on proteins and peptides of Homo sapiens origin (2 859 520 sequences; 980 631 860 residues). Search parameters are detailed in table legends. Identification required a minimum of two peptides for each protein and an Expect value of e0.05 unless otherwise indicated.
Results Identification of GCF Peptides. To determine a typical MS profile of GCF from inflamed sites, MALDI-TOF spectra of GCF were acquired from 30 individual sites, and an average spectrum of each was produced (Figure 1, Table 1, sample set 1). Four major peaks above m/z 10 000 in Figure 1B were putatively assigned to hemoglobin R and β chains and to calgranulin A and B based on their mass and their correspondence to major proteins identified from in-gel digests (see bands M10, M11, M12 in Figure 3, Table 3 and Table 4). LIFT MS/MS was attempted on peaks
