Mariaimmacolata Preianò1, Daniela Falcone1, Giuseppina Maggisano1, Tiziana Montalcini2, Michele Navarra3, Sergio Paduano1, Rocco Savino1, Rosa Terracciano4. 1. Department of Health Sciences, University "Magna Græcia" Catanzaro, University Campus, Europa Avenue, 88100 Catanzaro, Italy. 2. Department of Medical and Surgical Sciences, University "Magna Græcia" Catanzaro, University Campus, Europa Avenue, 88100 Catanzaro, Italy. 3. Department of Drug Sciences and Products for Health, University of Messina, Viale Annunziata, 98168 Messina, Italy. 4. Department of Health Sciences, University "Magna Græcia" Catanzaro, University Campus, Europa Avenue, 88100 Catanzaro, Italy. Electronic address: terracciano@unicz.it.
Abstract
BACKGROUND: Gingival 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. METHODS: GCF 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. RESULTS: Trifluoroacetic 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). CONCLUSION: An 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.
BACKGROUND: Gingival 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. METHODS: GCF 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. RESULTS:Trifluoroacetic 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). CONCLUSION: An 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.
Authors: M Castagnola; E Scarano; G C Passali; I Messana; T Cabras; F Iavarone; G Di Cintio; A Fiorita; E De Corso; G Paludetti Journal: Acta Otorhinolaryngol Ital Date: 2017-04 Impact factor: 2.124