Targeted Proteomics Guided by Label-free Quantitative Proteome Analysis in Saliva Reveal Transition Signatures from Health to Periodontal Disease

Abstract

Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. Here we carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva ( = 67, including individuals with health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort ( = 82). The LFQ platform led to the discovery of 119 proteins with at least 2-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 functionally related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases with maximum area under the receiver operating curve >0.97 (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1). This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum leap brought into the field of periodontal diagnostics by this study is the application of the biomarker discovery-through-verification pipeline, which can be used for validation in further cohorts.

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

“…To date, antibody‐based immunoassays are the main source for protein quantification using antibodies specifically targeting a protein of interest . Since antibodies might differ in sensitivity and specificity, might not detect all molecular forms of a protein or might not be available, MS has been recognized as a newer “gold standard” for the identification and analysis of individual proteins in complex biological samples . In this context, quantitative shotgun proteomics is a progressing field to identify potential biomarkers for periodontal disease in a time‐saving and high‐throughput approach with over 5000 proteins being discovered in saliva so far .…”

“…This might also explain the rather irregular regulation pattern of the biomarkers during the induction and resolution phases when measured by either IFMA or ELISA. In this context, novel targeted proteomics approaches could be used to overcome these drawbacks . An LFQ‐driven selected reaction monitoring (SRM) approach could design novel “anti‐peptide” antibodies against protein‐specific peptide sequences identified before by LFQ proteomics, for example, the top three ionizing peptides as used in this study.…”

“…Liquid chromatography‐mass spectrometry (LC‐MS)‐based proteomic approaches have demonstrated their valuable contribution in dissecting the human salivary proteome in health and disease . Using LC‐MS‐based proteomics for analyzing saliva constitutes a sensitive large‐scale application capable of identifying and quantifying various disease‐associated proteins simultaneously in a given sample . More than 5000 proteins were quantified in saliva, but further characterization and validation of these putative proteins is challenging due to the lack of availability of antibodies with high specificity for all of those …”

“…Further, although most of the assays are evaluated for a vast variety of sample matrixes, the complexity of saliva has to be taken into account . The implementation of a biomarker‐based assay into the routine clinical assessment requires certain features regarding assay performance in a clinical setting such as simple layout, time‐ and resource‐saving, non‐invasive, high throughput, highly sensitive, and specific …”

Effect of Chewing on the Expression of Salivary Protein Composition: A Systematic Review

“…More than 5000 proteins have been reported in saliva which have several biological and antimicrobial functions that influence numerous aspects of the oral health . Majority (90%) of these proteins originate from the three major salivary glands (the parotid, submandibular, and sublingual glands).…”