Assessment of pre-analytical and analytical variables affecting peptidome profiling of gingival crevicular fluid by MALDI-TOF mass spectrometry

• A standardized MALDI-TOF profiling of gingival fluid from healthy gums is proposed.
• A 30 s sampling time by paper points or paper strips does not affect MALDI spectra.
• Elution by trifluoroacetic acid generates richer spectra than elution by acetic acid.
• Robust MALDI spectra with CV < 10% from minute volume of fluid were generated.
• Standard signatures of gingival fluid may serve for the maintenance of healthy gums.

BackgroundGingival crevicular fluid (GCF) may be a source of new biomarkers of periodontitis/gingivitis. However, the minute volumes of GCF harvested in healthy sites are a serious drawback. We evaluated how pre-analytical and analytical variables concerning GCF collection and processing, could significantly influence quality and reproducibility of MALDI-TOF profiles.MethodsGCF was collected from healthy sites. The use of paper strips vs paper points was compared. Peptides and proteins were extracted by centrifugal elution in different solutions, at different accelerations, with and without protease inhibitor cocktail (PIC). Finally, we evaluated how matrix composition and matrix/sample volume ratio affect the reproducibility of MALDI-TOF profiles.ResultsTrifluoroacetic acid elution generated richer gingival fingerprints compared to acetic acid, independently of the collection device. Centrifugation speed and PIC supplementation did not change GCF profiles. A fine modulation of matrix composition and matrix/sample volume ratio resulted in a satisfactory reproducibility (CV less than 10% for peak area and signal-to-noise ratio).ConclusionAn optimized procedure, enabling generation of reproducible MALDI-TOF profiles from limited volume of GCF, is proposed. These fingerprints may serve as reference for future studies oriented to the maintenance and preservation of good gingival status and to discovery biomarkers of periodontitis/gingivitis.