Clinical Oral Investigations

, Volume 22, Issue 3, pp 1375–1384 | Cite as

A new albumin-depletion strategy improves proteomic research of gingival crevicular fluid from periodontitis patients

  • Sarah Batschkus
  • Goekhan Cingoez
  • Henning Urlaub
  • Nicolai Miosge
  • Christian Kirschneck
  • Philipp Meyer-Marcotty
  • Christof Lenz
Original Article



Gingival crevicular fluid (GCF), the inflammatory infiltrate within the crevicular sulcus, is of great importance for diverse processes in the oral cavity and has a high impact in oral sciences. It is assumed to serve as a source of biomarkers for systemic or periodontal diseases and mediators of orthodontic tooth movement. In order to characterize the protein content of the GCF in an unbiased and complete approach, we employed mass spectrometry (MS), which allows not only the identification, but also the quantification of these proteins. In samples obtained from patients suffering from periodontitis, this method is often limited due to the presence of highly abundant serum albumin deriving from serum. The aim of this investigation was to employ a protein precipitation procedure for the efficient depletion of serum albumin from GCF samples.

Materials and methods

GFC samples collected from five adult periodontitis patients were fractionated by trichloroacetic acid/acetone precipitation and the resulting soluble and pelleted fractions were analyzed by SDS-PAGE and high-resolution mass spectrometry.


Trichloroacetic acid/acetone precipitation was successfully employed as a protein precipitation procedure for the efficient depletion of serum albumin from GCF samples. Careful analysis revealed that the precipitation step reduced the serum albumin content efficiently, and increased subsequent protein identifications by 32%. Three hundred seventeen proteins could only be identified with this new approach.


The increased coverage of the GCF proteome will help improve our understanding of molecular mechanisms in the periodontium during pathogenesis of periodontitis.

Clinical relevance

Our new albumin depletion strategy combined with high-resolution mass spectrometry can be used to effectively monitor the molecular signals of the periodontium.


Gingival crevicular fluid Mass spectrometry Serum albumin Periodontitis Orthodontic tooth movement 



There was no grant support or funding for this study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was by the responsible ethics committee (University of Goettingen) with protocol number 23/7/15.

Informed consent

Informed consent was obtained from all individual participants included in this study.

Supplementary material

784_2017_2213_MOESM1_ESM.pdf (1.3 mb)
Supplementary Table 1 (PDF 1323 kb)
784_2017_2213_MOESM2_ESM.pdf (1.2 mb)
Supplementary Table 2 (PDF 1246 kb)


  1. 1.
    Rody WJ Jr, Holliday LS, McHugh KP et al (2014) Mass spectrometry analysis of gingival crevicular fluid in the presence of external root resorption. Am J Orthod Dentofac Orthop 145(6):787–798. CrossRefGoogle Scholar
  2. 2.
    Carneiro LG, Venuleo C, Oppenheim FG et al (2012) Proteome data set of human gingival crevicular fluid from healthy periodontium sites by multidimensional protein separation and mass spectrometry. J Periodontal Res 47(2):248–262. CrossRefPubMedGoogle Scholar
  3. 3.
    Ngo LH, Darby IB, Veith PD et al (2013) Mass spectrometric analysis of gingival crevicular fluid biomarkers can predict periodontal disease progression. J Periodontal Res 48(3):331–341. CrossRefPubMedGoogle Scholar
  4. 4.
    Uitto V-J (2003) Gingival crevice fluid—an introduction. Periodontol 2000(31):9–11CrossRefGoogle Scholar
  5. 5.
    Embery G, Waddington R (1994) Gingival crevicular fluid: biomarkers of periodontal tissue activity. Adv Dent Res 8(2):329–336CrossRefPubMedGoogle Scholar
  6. 6.
    Alfano MC (1974) The origin of gingival fluid. J Theor Biol 47(1):127–136CrossRefPubMedGoogle Scholar
  7. 7.
    Carneiro LG, Nouh H, Salih E (2014) Quantitative gingival crevicular fluid proteome in health and periodontal disease using stable isotope chemistries and mass spectrometry. J Clin Periodontol 41(8):733–747. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ren Y, Vissink A (2008) Cytokines in crevicular fluid and orthodontic tooth movement. Eur J Oral Sci 116(2):89–97. CrossRefPubMedGoogle Scholar
  9. 9.
    Vishwakarma A, Sharpe P, Shi S et al (2014) Stem cell biology and tissue engineering in dental sciences. Elsevier Science, AmsterdamGoogle Scholar
  10. 10.
    Wilmes P, Heintz-Buschart A, Bond PL (2015) A decade of metaproteomics: where we stand and what the future holds. Proteomics 15(20):3409–3417. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bao K, Belibasakis GN, Selevsek N et al (2015) Proteomic profiling of host-biofilm interactions in an oral infection model resembling the periodontal pocket. Sci Rep 5:15999. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Barbieri G, Solano P, Alarcon JA et al (2013) Biochemical markers of bone metabolism in gingival crevicular fluid during early orthodontic tooth movement. Angle Orthod 83(1):63–69. CrossRefPubMedGoogle Scholar
  13. 13.
    Kereshanan S, Stephenson P, Waddington R (2008) Identification of dentine sialoprotein in gingival crevicular fluid during physiological root resorption and orthodontic tooth movement. Eur J Orthod 30(3):307–314. CrossRefPubMedGoogle Scholar
  14. 14.
    Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422(6928):198–207. CrossRefPubMedGoogle Scholar
  15. 15.
    Andrews GL, Simons BL, Young JB et al (2011) Performance characteristics of a new hybrid triple quadrupole time-of-flight tandem mass spectrometer. Anal Chem 83(13):5442–5446. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372. CrossRefPubMedGoogle Scholar
  17. 17.
    Tsuchida S, Satoh M, Umemura H et al (2012) Proteomic analysis of gingival crevicular fluid for discovery of novel periodontal disease markers. Proteomics 12(13):2190–2202. CrossRefPubMedGoogle Scholar
  18. 18.
    Bostanci N, Heywood W, Mills K et al (2010) Application of label-free absolute quantitative proteomics in human gingival crevicular fluid by LC/MS E (gingival exudatome). J Proteome Res 9(5):2191–2199. CrossRefPubMedGoogle Scholar
  19. 19.
    Silva-Boghossian CM, Colombo APV, Tanaka M et al (2013) Quantitative proteomic analysis of gingival crevicular fluid in different periodontal conditions. PLoS One 8(10):e75898. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Armitage GC (2000) Development of a classification system for periodontal diseases and conditions. Northwest Dent 79(6):31–35PubMedGoogle Scholar
  21. 21.
    Chen Y-Y, Lin S-Y, Yeh Y-Y et al (2005) A modified protein precipitation procedure for efficient removal of albumin from serum. Electrophoresis 26(11):2117–2127. CrossRefPubMedGoogle Scholar
  22. 22.
    Neuhoff V, Arold N, Taube D et al (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9(6):255–262. CrossRefPubMedGoogle Scholar
  23. 23.
    Schmidt C, Lenz C, Grote M et al (2010) Determination of protein stoichiometry within protein complexes using absolute quantification and multiple reaction monitoring. Anal Chem 82(7):2784–2796. CrossRefPubMedGoogle Scholar
  24. 24.
    Shilov IV, Seymour SL, Patel AA et al (2007) The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra. Mol Cell Proteomics 6(9):1638–1655. CrossRefPubMedGoogle Scholar
  25. 25.
    Nesvizhskii AI, Keller A, Kolker E et al (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75(17):4646–4658CrossRefPubMedGoogle Scholar
  26. 26.
    Liu H, Sadygov RG, Yates JR (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76(14):4193–4201. CrossRefPubMedGoogle Scholar
  27. 27.
    Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57. CrossRefGoogle Scholar
  28. 28.
    Huang DW, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37(1):1–13. CrossRefGoogle Scholar
  29. 29.
    Delaville M, Delaville G, Delaville J (1954) Solubility characteristics of the albumin fraction of blood serum in ethyl alcohol-trichloroacetic acid solutions; application to the determination of the various protein fractions of serum (Caractere de solubilite de la fraction albuminique du serum sanguin dans les solutions d'ethanol trichloracetique; application au dosage des diverses fractions proteiques du serum). Ann Biol Clin (Paris) 12(5–6):320–323Google Scholar
  30. 30.
    Levine S (1954) Solubilization of bovine albumin in nonaqueous media. Arch Biochem Biophys 50(2):515–517CrossRefPubMedGoogle Scholar
  31. 31.
    Choi Y-J, Heo S-H, Lee J-M et al (2011) Identification of azurocidin as a potential periodontitis biomarker by a proteomic analysis of gingival crevicular fluid. Proteome Sci 9(1):42. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Eley BM, Cox SW (1992) Cathepsin B/L-, elastase-, tryptase-, trypsin- and dipeptidyl peptidase IV-like activities in gingival crevicular fluid: a comparison of levels before and after periodontal surgery in chronic periodontitis patients. J Periodontol 63(5):412–417. CrossRefPubMedGoogle Scholar
  33. 33.
    Travan S, Li F, D'Silva NJ et al (2013) Differential expression of mitogen activating protein kinases in periodontitis. J Clin Periodontol 40(8):757–764. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Capelli J, Kantarci A, Haffajee A et al (2011) Matrix metalloproteinases and chemokines in the gingival crevicular fluid during orthodontic tooth movement. Eur J Orthod 33(6):705–711. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department for Orthodontics, Center for Dentistry, Oral Medicine and Craniomaxillofacial Surgery, University Medical CenterGeorg August UniversityGöttingenGermany
  2. 2.Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass Spectrometry GroupGoettingenGermany
  3. 3.Institute of Clinical Chemistry, Bioanalytics GroupUniversity Medical Center GoettingenGoettingenGermany
  4. 4.Tissue Regeneration Group, Department of ProsthodonticsUniversity Medical Center GoettingenGoettingenGermany
  5. 5.Department of OrthodonticsUniversity Medical Center RegensburgRegensburgGermany

Personalised recommendations