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

<jats:sec><jats:title>Purpose</jats:title><jats:p>This study aims to validate label‐free quantitative proteomics (LFQ) against antibody‐based methods for quantifying established periodontal disease biomarkers in saliva.</jats:p></jats:sec><jats:sec><jats:title>Experimental Design</jats:title><jats:p>In an experimental gingivitis model, healthy volunteers (<jats:italic>n</jats:italic> = 10) provide saliva at baseline (d0), during the induction (d7, d14, d21) and resolution (d35) of gingival inflammation (total <jats:italic>n</jats:italic> = 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.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>LFQ detects significantly (<jats:italic>p</jats:italic> < 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 (<jats:italic>r</jats:italic> = 0.36), myeloperoxidase (<jats:italic>r</jats:italic> = 0.39), polymorphonuclear leukocyte elastase (<jats:italic>r</jats:italic> = 0.33), and TIMP‐1 (<jats:italic>r</jats:italic> = −0.24).</jats:p></jats:sec><jats:sec><jats:title>Conclusion and Clinical Relevance</jats:title><jats:p>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.</jats:p></jats:sec>