Analysis of proteins in human gingival crevicular fluid by mass spectrometry.
OBJECTIVE
Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry.


METHODS
Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting.


RESULTS
One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting.


CONCLUSIONS
Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.
Department of Periodontology and Endodontology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan. kido@dent.tokushima-u.ac.jp
J
J
Kido
M
M
Bando
Y
Y
Hiroshima
H
H
Iwasaka
K
K
Yamada
N
N
Ohgami
T
T
Nambu
M
M
Kataoka
T
T
Yamamoto
Y
Y
Shinohara
I
I
Sagawa
T
T
Nagata
D000328:Adult; D000368:Aged; D023181:Antimicrobial Cationic Peptides / Q000032:analysis*; D015153:Blotting, Western; D016022:Case-Control Studies; D002570:Ceruloplasmin / Q000032:analysis; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005883:Gingival Crevicular Fluid / Q000737:chemistry* / Q000201:enzymology; D005982:Glutathione Transferase / Q000032:analysis; D024981:Glycogen Phosphorylase / Q000032:analysis; D006801:Humans; D008297:Male; D008875:Middle Aged; D010514:Periodontal Pocket / Q000201:enzymology / Q000378:metabolism*; D010518:Periodontitis / Q000175:diagnosis*; D010736:Phosphoglycerate Mutase / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D052243:Resistin / Q000032:analysis; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000379:methods*
D023181:Antimicrobial Cationic Peptides; D011506:Proteins; D052243:Resistin; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins; C110294:S100A1 protein; C089242:S100A7 protein, human; D002570:Ceruloplasmin; D024981:Glycogen Phosphorylase; D005982:Glutathione Transferase; D010736:Phosphoglycerate Mutase