Top down proteomic analysis of gingival crevicular fluid in deciduous, exfoliating and permanent teeth in children

Highlights

• GCF was collected from deciduous, exfoliating and permanent teeth from 20 children.

• Peptide/proteins content was characterized by a Top-Down MS proteomic approach.

• The data help to understand complex biological processes like teeth eruption.

• Data are available via ProteomeXcharge with identifier PXD016010 and PXD016049.

Abstract

Gingival Crevicular Fluid (GCF), a plasma-derived exudate present in the gingival crevice was collected from deciduous, exfoliating and permanent teeth from 20 children (60 samples) with the aim to characterize and quantify by a mass spectrometry based top-down proteomic approach, the peptide/proteins in the fluid and verify possible variations occurring during the exfoliating process.

The results obtained confirmed the presence in GCF of α-Defensins 1–4, Thymosin β4 and Thymosin β10, as described in previous works and revealed the presence of other interesting peptides never described before in GCF such as specific fragments of α-1-antitrypsin, α-1-antichymotrypsin; fragments of Thymosin β4 and Thymosin β10; Fibrinopeptide A and its fragments and Fibrinopeptide B; S100A8 and S100A9, LVV Hemorphin-7 (hemoglobin chain β fragment), as well as some other peptides deriving from α and β subunits of hemoglobin. Statistical analysis evidenced different levels in 5 proteins/peptides in the three groups. Our study demonstrate that an in-depth analysis of a biological fluid like GCF, present in small amount, can provide useful information for the understanding of different biological processes like teeth eruption. Data are available via ProteomeXchange with identifier PXD016010 and PXD016049.

Significance

GCF due to his site-specific nature has a great potential in containing factors that are specific for action at a given site and might have diagnostic value to detect qualitative and quantitative variations of proteins/peptides composition linked to physiological or pathological conditions.

Introduction

Human Gingival Crevicular Fluid (GCF) is a plasma-derived exudate present in the gingival crevice of the periodontal pocket, between teeth and the surrounding gingival tissue [1]. The constituents of the fluid arise from the host as well as from microorganisms resident in the subgingival and supragingival plaque. Contribution from the host includes molecules deriving from blood and from cells and tissues of the periodontium. In fact, the junctional epithelium is infiltrated by neutrophils, mononuclear cells and lymphocytes [2]. GCF composition varies between healthy and diseased periodontium conditions [3]. Important host-derived constituents in GCF are markers of inflammation including enzymes, cytokines, and interleukins. Further, products of tissue breakdown can also be detected [4].

The major attraction of GCF for its possible diagnostic use is the site-specific nature of the sampling. Indeed, analysis of GCF collected from distinct sites/teeth could offer the potential of diagnostic information for individual sites and teeth. In previous studies, our group has been involved in the analysis of the GCF fluid in adults with low-resolution top-down proteomic platform [5,6]; while in this study we have deepened the analysis of proteins and peptides in children's GCF with a high resolution top-down pipeline. GCF from deciduous and permanent teeth has been already analyzed in children [1,7] and biomarkers of bone and tissue metabolism associated with orthodontic tooth movement, neutrophil-related proteins and immunoglobulins were identified. However, we have a limited understanding of the physiological changes occurring in GCF biochemical composition when deciduous teeth are replaced by permanent teeth, and little information is available on GCF of exfoliating teeth [8].

Previous studies reported the changing of gingival tissues surrounding permanent and deciduous teeth during the replacement [9,10]. Tooth eruption is a complex process, and little is known about the mechanism of its control [11]. The main hypothesis is that the periodontal ligament promotes eruption through the shrinking and cross-linking of collagen fibers and the contraction of the fibroblasts [12].

Areas of tension and compression are generated in the soft tissues surrounding unerupted teeth by the distribution of bite forces through the jaws. These patterns of tension and compression are further proposed to result in patterns of bone resorption and deposition that lift the tooth into the mouth [13]. This theory is based on Wolff's Law, which is the long-established idea that bone changes shape in accordance with the forces applied [14]. In deciduous dentition, all these processes are active especially in 10 yrs. old children where deciduous dentition is near to be replaced by permanent dentition. The aim of this study was to analyze GCF protein composition in deciduous, exfoliating and permanent teeth in children and check if variations occur during the exfoliating process by a top-down strategy based on a high-resolution nano-HPLC-ESI-MS platform.

High-resolution mass spectrometry equipped with micro/nano-ESI source and LC devices is an important tool for the in deep and accurate molecular characterization of biological fluids available in few amounts such us GCF. Furthermore, the top-down proteomics approach that explores the intact proteins and the peptides naturally occurring in the sample, minimizing, as much as possible, any alteration during pretreatment [[15], [16], [17]] is a powerful analytical platform for investigating both the qualitative and quantitative variations of GCF proteins/peptides composition linked to physiological or pathological conditions and for disclosing proteoforms and PTMs possible alterations [18,19].

Top-down approach, as demonstrated in papers previously published by our group [16,17], provides univocal information and instantaneous images on the protein content and modification of the fluid under examination at the moment in which it is withdrawn, and allows a direct qualitative-quantitative comparison in samples originated from different sites as described in this study.