The 21-day experimental gingivitis model is an established noninvasive model in humans for investigating the induction and resolution of inflammation in response to increasing bacterial accumulation. From its inception in 1965,1,2
this model of inflammation has been used to assess how drugs,(3
) bioactive compounds in dentifrices4−6
and environmental toxins, such as tobacco smoke,7,8
affect the development of inflammation. The model is designed to enable the study of both the induction and resolution of inflammation, which can also be assessed over a relatively short period of time (35 days) and in a controlled manner.
The model involves periodontally and systemically healthy volunteers, who do not brush specified test teeth in one sextant of the dentition, but continue to brush the remaining control teeth over a 21 day period. Accidental brushing of test teeth is prevented by the use of a vinyl shield (mouth guard) to cover them during cleaning. On day 21, the accumulated plaque on all teeth is removed by a trained hygienist and study participants resume their normal oral hygiene practices. Clinical assessment of plaque accumulation and gingival inflammation are made at baseline, day 7, 14, 21 on test and control teeth, following plaque accumulation on test teeth, and normal plaque removal from control teeth. Assessments are repeated again on day 35, following 14 days of resolution of inflammation at test sites, and maintenance of health at control sites. When performed by trained and calibrated experts, these measures show consistent increases in plaque-induced inflammation up to day 21 followed by a return to baseline, preinflammation levels at day 35.9,10
However none of these assessments could potentially form the basis for a predictor of disease as they are all measures of existing inflammation or inducers of inflammation.
A further and more objective assessment that can be made during the experimental gingivitis is that of the volume and composition of gingival crevicular fluid (GCF) and at individual sites around individual teeth. This fluid flows from the crevice between the tooth and the gum and comprises both a serum transdate(11
) and tissue exudates.3,10,12−14
During inflammation, the volume and flow of this fluid increases,(1
) and this alone can be used to demonstrate induction and/or presence of gingivitis and periodontitis. However, due to its very nature, as a mixed serum transudate and tissue exudate, GCF carries with it proteins from the crevice. Thus its composition also provides a biological and pathological fingerprint of the various physiological and biochemical processes occurring within the gingival tissues. In 1980, Novaes et al.(15
) demonstrated that the total protein present in GCF increased with increasing severity of periodontitis, although this was probably due to increases in GCF volume. By 1985, Lamster et al.(16
) had measured differences in lactate dehydrogenase, beta-glucuronidase and arylsulfatase activity in GCF collected from experimental gingivitis sites and demonstrated increases in these biomarkers over the course of the 21 day study. Since then, studies have targeted particular cytokines,3,10,12−14,17,18
antibacterial peptides and proteins(19
) and many more individual proteins and peptides,9,20−22
revealing increases in pro-inflammatory species during disease. Studies have also examined the effect of diseases, such as type I diabetes mellitus,(23
) analysis of many anti-inflammatory or antimicrobial compounds, and environmental conditions, such as smoking,(8
) on experimental gingivitis.
Recently, proteomics techniques have greatly increased understanding of the composition of GCF. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF MS) has been used to examine small proteins and peptides (2.5−30 kDa) from healthy volunteers and gingivitis patients.(24
) That study employed a targeted approach to investigate neutrophil defensins. Unfractionated GCF samples were also examined by matrix assisted laser desorption/ionization (MALDI)-TOF MS, for neutrophil defensins.(25
) Similarly, Pisano et al(26
) used electrospray ionization(27
) (ESI)−MS to examine the acid-soluble protein content of GCF from healthy volunteers. They, too, identified the neutrophil defensins, along with cystatin A, statherin and serum albumin. Only in the last year have tandem mass spectrometry (MS/MS) techniques been employed in the examination of GCF. Ngo et al.(28
) used both MALDI-TOF/TOF and ESI−MS/MS to identify 66 proteins that had been separated by gel electrophoresis from a single patient, who had a history of periodontal disease but who was in the maintenance phase following treatment. Proteins identified included a number of serum-derived proteins, such as albumin, complement and transferrin. Other proteins that were present included: antimicrobial proteins calgranulin A and B; amylase; cytatin S; and Histone H4. Bostanci et al.(29
) used a LC−MSE
label-free quantitative technique to identify 154 proteins from either healthy volunteers or patients with aggressive periodontitis. Bostanci et al.(29
) identified a wide range of proteins including those mentioned already and a large number of keratins, immunoglobulins, and other intracellular proteins. They also potentially identified a number of bacterial, fungal and viral proteins.
The majority of mass spectrometry-based proteomics is performed using the bottom-up(30
) liquid chromatography tandem mass spectrometry (LC−MS/MS) approach. Generally, proteins are digested with trypsin prior to online LC separation. As the peptides elute into the mass spectrometer, they are fragmented typically by collision induced dissociation(31
) (CID) resulting in b
) The MS/MS spectra are searched against protein databases by use of algorithms (e.g., Mascot,(33
)) which match the data to theoretical spectra from in-silico digests of proteins. Quantitative proteomics by mass spectrometry can use several methods. Two of the most popular are stable isotope labeled amino acids in cell culture(36
) (SILAC) and isobaric tags for relative and absolute quantitation (iTRAQ).(37
) In SILAC, cells are grown in media lacking essential amino acids. The media is supplemented with heavy or light amino acids. The labeled and unlabeled peptides elute together and the intensity of the peptide ions can be compared. SILAC is not suitable for clinical samples. A common approach for quantitation of biological fluids(38
) and tissue samples(39
) is postdigestion labeling with iTRAQ(40
) labels. iTRAQ labels consist of a reporter ion with a m
value of between 113 and 121 and a balance mass (191−183 Da) such that all labeled peptides of the same sequence have the same nominal mass shift (Δ304 Da). Test and control samples are treated with separate iTRAQ labels postdigestion and combined prior to MS analysis. When the peptide ions are fragmented with CID, the reporter ions are cleaved from the peptide and detected in the mass spectrometer. The intensity of the reporter ions can then be quantified. Quantitation with iTRAQ, allows for the analysis up to eight different treatment groups(41
) simultaneously. One of the disadvantages of performing CID in ion traps is the instability of ions with m
values approximately 1/3 that of the parent ion,(42
) that is, fragment ions with m
less than 1/3 of the precursor ions are not detected. iTRAQ reporter ions have m
113−121 and therefore ion trap CID of iTRAQ labeled peptides often does not result in quantification. The recent introduction of pulsed-Q dissociation(43
) (PQD) has allowed for the analysis of iTRAQ labeled samples in linear ion trap mass analysers. PQD excites ions with a high amplitude Q value; Q is a resonance excitation pulse. When the ions are excited, they collide with neutral gas molecules in a similar manner to CID. The activation profile applied in PQD is different to that of CID meaning that low mass ions are stable and can be detected.
To date, the proteomic profile of GCF during the active induction and subsequent resolution of inflammation under controlled conditions using the 21 day experimental gingivitis model has not been described. Herein we describe the quantitative analysis of GCF, collected from volunteers undergoing experimental gingivitis, by LC−MS/MS using Fourier transform ion cyclotron resonance(44
) (FT-ICR) MS and iTRAQ isobaric mass tags, to establish a profile of changes in proteins in healthy young volunteers that may be used to compare to the inflammatory response in other subsets of the population, such as those predisposed to periodontitis.