Label‐Free Quantitative Proteomics versus Antibody‐Based Assays to Measure Neutrophil‐Derived Enzymes in Saliva

Abstract

Purpose: This study aims to validate label-free quantitative proteomics (LFQ) against antibody-based methods for quantifying established periodontal disease biomarkers in saliva. Experimental Design: In an experimental gingivitis model, healthy volunteers (n = 10) provide saliva at baseline (d0), during the induction (d7, d14, d21) and resolution (d35) of gingival inflammation (total n = 50). Biomarker levels are analyzed by LFQ and time-resolved immunofluorometric assay (IFMA) or enzyme-linked immunosorbent assay (ELISA). Molecular matrix metalloproteinase (MMP)-8 forms are assessed by Western blot (WB) analysis. Results: LFQ detects significantly (p < 0.05) elevated MMP-8 (d21vsd7, d35vsd7) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 (d35vsd7). Latent MMP-8 (70-80 kDa) is present (d0-d35), but not active MMP-8 (50-60 kDa). LFQ and immunoassay data significantly correlate for MMP-8 (r = 0.36), myeloperoxidase (r = 0.39), polymorphonuclear leukocyte elastase (r = 0.33), and TIMP-1 (r = −0.24).Conclusion and Clinical Relevance: LFQ can quantify enzyme levels in saliva, however lacks the ability to measure enzymatic activity. WB analysis reveals that MMP-8 may not be activated during induction of gingival inflammation. Significant but weak correlations between IFMA or ELISA and LFQ suggest a limited capacity of available antibodies to reliably quantify salivary biomarkers for periodontal diseases. Novel "anti-peptide" antibodies designed by newer targeted mass spectrometry-based approaches can help to overcome these drawbacks.

Salivary Biomarkers for Dental Caries Detection and Personalized Monitoring

“…All the saliva samples were screened for 44 candidate biomarkers (cytokines, chemokines, growth factors, matrix metalloproteinases, a metallopeptidase inhibitor, proteolytic enzymes, and selected oral bacteria). These biomarkers were selected as a multitargeted approach to addressing different immune responses, which were, in part, already described as correlating with oral diseases [27][28][29][30][31][32][33]. Patient enrollment and saliva collection took place from September 2018 to April 2019 and were approved by the local Swiss ethics committee (BASEC-no.…”

“…This may be achieved by validated combinations of host-derived salivary biomarkers and key pathogens [ 23 , 24 , 25 , 26 ]. Common biomarker panels include cytokines (such as the interleukins IL-6 and IL-1β), matrix metalloproteinases (MMP-8 and MMP-9), a metallopeptidase inhibitor (TIMP-1) and the presence of periodontal pathogens (e.g., Porphyromonas gingivalis , Treponema denticola , Tanerella forsythia, Fusobacterium nucleatum , Campylobacter rectus , and Prevotella intermedia ) [ 27 , 28 , 29 , 30 , 31 , 32 , 33 ].…”

Salivary proteotypes of gingivitis tolerance and resilience

“…The current study analysed 50 saliva samples and included "fast" responders (mean age: 22.0 years; F:M: 3:2), "slow" responders (mean age: 24.0 years; F:M: 3:2) and total sample ("fast" and "slow" responders combined, mean age: 23.1 years; F:M: 6:4). The mean gingival inflammation (MGI) and plaque (TQHPI) scores for "fast" and "slow" responders during the induction and resolution phases F I G U R E 1 Schematic representation of the study design from saliva collection to protein discovery using label-free quantitative (LFQ) proteomics of gingival inflammation are summarized in Figure 2 and Supporting File 2 (Silbereisen, Alassiri, et al, 2019).…”

Proteomic analysis of human immunodeficiency virus and periodontitis

“…However, the detailed mechanism by which systemic disease is related to periodontal disease remains unclear. MS-based approaches can quantify enzyme levels in biological fluids, but they lack the capacity to measure their enzymatic activity [11]. The mechanistic aspects underlying the disease state are unknown, although the onset of periodontal disease is reportedly triggered by bacterial periodontal infection.…”