Untargeted Salivary Metabolomics and Proteomics: Paving the Way for Early Detection of Periodontitis
Abstract
1. Introduction
2. Materials and Methods
3. Periodontitis: Pathogenesis and Early Diagnosis
3.1. Early Detection and Salivary Diagnosis of Periodontitis
3.2. Saliva Compared with Gingival Crevicular Fluid
4. Salivary Samples: The Importance of the Correct Collection and Preparation
5. Salivary Metabolomics: Techniques and Applications
6. Salivary Proteomics: Techniques and Applications
7. Targeted and Untargeted Approaches in Biomarker Discovery
8. Analytical Technologies and Data Interpretation
9. Challenges and Limitations
10. Future Perspectives
11. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Metabolomics Sequencing Techniques | Methodologies | Strengths | Limitations | Potential Role in Early Diagnosis of Periodontitis | References |
---|---|---|---|---|---|
NMR Spectroscopy | Detects nuclear magnetic resonance signals (commonly 1H, 13C); non-destructive and quantitative. | - High reproducibility - Non-destructive - Absolute quantification | - Low sensitivity - Signal overlap - Costly instruments | Reliable for saliva metabolite fingerprinting and longitudinal studies. | [42,46,53,54] |
GC-MS | Separates volatile/derivatized compounds; detects by MS. | - High sensitivity - Good for small molecules - Established protocol | - Requires derivatization - Limited to volatile analytes | Useful for SCFAs and volatile microbial metabolites. | [55,56,57] |
LC-MS | Separates polar/non-polar metabolites in liquid phase; detects ions by MS. | - Broad coverage - High sensitivity - Versatile | - Ion suppression - Requires complex QC | Gold standard for untargeted saliva metabolomics. | [58,59] |
FTIR Spectroscopy | Measures infrared absorption of molecular bonds; generates spectral fingerprints. | - Rapid, label-free - Minimal preparation | - Lower specificity - Mostly qualitative - Sensitive to water | Distinguishes healthy from diseased saliva via biochemical fingerprinting. | [16,60,61] |
PAS (Photoacoustic Spectroscopy) | Measures acoustic waves generated after absorption of modulated light; used to assess biochemical changes. | - Non-invasive - High sensitivity to molecular vibrations - Can be miniaturized | - Lower metabolite specificity - Limited commercial systems - Sensitive to water | Emerging tool for real-time screening of periodontal changes in saliva. | [12,62,63] |
Biomarkers | Molecular Categories | Biological Pathways | Functions and Significance | References |
---|---|---|---|---|
Acetic, acetoacetic, butyric, citric, formic, isovalerate, formate, glycolic, lactic, propionic, and pyruvic acids. | SCFAs | They result from the fermentation of sugars and proteins from anaerobic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia), and some of them, like formate, may result from purine breakdown under oxidative stress. | Inducing apoptosis of gingival epithelial cells, impairing fibroblast function, suppressing immune cells, promoting dysbiosis and neutrophil recruitment, and decreasing pH. | [64,66,67,68,69,70,71] |
L-alanine, d-glutamic acid, l-valine, l-methionine, l-threonine, l-leucine, isoleucine, l-tyrosine, and l-phenylalanine. | Amino acids | They are energy sources for anaerobic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia, and they result from the inflammation and tissue damage process. | Markers of tissue degradation, elevated in inflammation, reflecting the host’s immune response involved in energy metabolism, and bacteria. | [64,72,73] |
Taurine, ethanolamine, dimethylamine, and methylamine. | Amines | They are released by the host immune system. | Antioxidant effect, in particular, Taurine. Maintaining cellular homeostasis under inflammatory conditions. | [64,74,75,76] |
D-glucose | Sugar | They are an energetic source for periodontal bacteria. | Augmented energy demand of bacteria. | [64,77] |
Hypoxanthine | Purine nucleobase | It is a byproduct of purine degradation mediated by bacteria. | Its presence indicates periodontal pathogens’ activity. | [78] |
References | Study Design | Sample Size | Method of Analysis | Variation in Metabolomic Profile |
---|---|---|---|---|
Aimetti et al. [79] | Cross-sectional study | 32 cases, 22 controls | Nuclear Magnetic Resonance | Higher concentrations of acetate, γ-aminobutyrate, n-butyrate, succinate, trimethylamine, propionate, phenylalanine, and valine, and decreased concentrations of pyruvate and N-acetyl in GCP patients compared with controls. |
Rzeznic et al. [42] | Cross-sectional study | 26 cases, 25 controls | Nuclear Magnetic Resonance | Higher concentrations of short-chain fatty acids and lower concentrations of lactate, γ-amino-butyrate, methanol, and threonine in periodontitis. |
Romano et al. [80] | Cross-sectional study | 33 cases with GCP, 28 cases with GAgP; 39 controls. | Nuclear Magnetic Resonance | Higher concentrations of pyruvate, N-acetyl groups, and lactate, and higher levels of proline, phenylalanine, and tyrosine in GCP and GAgP patients compared with controls. |
Kim et al. [74] | Cross-sectional study | 129 cases, 92 controls. | Nuclear Magnetic Resonance | Higher concentrations of taurine, isovalerate, butyrate, and glucose in periodontitis. |
García-Villaescusa et al. [39] | Case-control study | 91 cases, 39 controls. | Nuclear Magnetic Resonance | Higher concentrations of caproate, isocaproate, butyrate, isovalerate, isopropanol, methanol, 4-aminobutyrate, choline, sucrose, sucrose-glucose-lysine, lactate-proline, lactate, and proline in periodontitis. |
Biomarker | Molecular Category | Biological Pathway | Function and Significance | Reference |
---|---|---|---|---|
Matrix metalloproteinase-8 | Enzyme | It is released by neutrophils and fibroblasts | Breaking down collagen | [95] |
Complement C3 | Protein part of the complement system | It is triggered by immune complexes | Enhancing recognition and clearance of periodontal pathogens by phagocytes, recruiting neutrophils and other immune cells to the gingival tissues, leading to chronic inflammation | [96,97] |
Profilin-1 | Actin-binding protein | Rho GTPase signaling → Profilin-1 → Actin polymerization → Cell movement and adhesion. | Regulating actin polymerization and cytoskeletal growth | [98,99,100] |
S100A8 | Pro-inflammatory mediator | S100A8/A9 → TLR4/RAGE → NF-κB → Inflammation | Calcium ion binding Cytokine activity (in inflammatory states) | [18,101,102,103] |
Fibrinogen | Glycoprotein | Fibrinogen → Thrombin cleavage → Fibrin → Clot formation & tissue scaffolding | Blood coagulation factor, acute-phase reactant, ligand for integrins (e.g., Mac-1, αIIbβ3), interacts with toll-like receptors (e.g., TLR4). | [96,104,105] |
Cystatin-SN | Cysteine protease inhibitor | Cystatin-SN → Inhibition of cathepsins/proteases → Protection of connective tissue & regulation of inflammation | Cysteine-type endopeptidase inhibitor activity, protease binding | [106,107,108] |
References | Study Design | Sample Size | Method of Analysis | Variation in the Proteomic Profile |
---|---|---|---|---|
Shin et al. [18] | Cross-sectional study | 36 cases, 36 controls | Mass spectrometry-based untargeted proteomics | S100A8 and S100A9 higher in periodontitis patients. |
Belstrøm et al. [87] | Cross-sectional study | 10 cases, 10 controls | Mass spectrometry-based untargeted proteomics | Proteins associated with innate immune response were higher in Periodontitis patients |
Bostanci et al. [107] | Cross-sectional study | 17 CP, 17 AgP; 16 controls | Mass spectrometry-based untargeted proteomics | Lactoferrin, lacritin, sCD14, Mucin 5B, and Mucin 7 down-regulated in AP and SLC4A1 was upregulated. Cystatin SN higher in controls |
Mertens et al. [109] | Cross-sectional study | 10 CP, 11 AgP; 12 controls. | Mass spectrometry-based untargeted proteomics. | Hemopexin, plasminogen, and α-fibrinogen were higher in periodontitis patients. |
Tang et al. [110] | Cross-sectional study | 16 cases, 17 controls. | Mass spectrometry-based untargeted proteomics. | Two peptide peaks had a lower level of intensity in the CP group, while the rest of the differentially expressed peptides had a higher level of intensity. |
Antezack et al. [111] | Cross-sectional study | 67 cases, 74 controls | Mass spectrometry-based untargeted proteomics | No identification of specific proteins |
Hartenbach et al. [112] | Cross-sectional study | 30 cases, 10 controls. | Mass spectrometry-based untargeted proteomics. | Salivary acidic proline-rich phosphoprotein, submaxillary gland androgen-regulated protein, histatin-1, fatty acid binding protein, thioredoxin, and cystatin-SA higher in periodontitis patients. |
Grant et al. [103] | Cross-sectional study | 10 MMP, 10 AP; 10 controls. | Mass spectrometry-based untargeted proteomics. | MMP9, S100A8, A1AGP, and pyruvate kinase higher in periodontitis patients. |
Casarin et al. [113] | Cross-sectional study | 12 cases, 13 controls | Mass spectrometry-based untargeted proteomics | GHG1, CSTB, KRT9, SMR3B, IGHG4, and SERPINA1 were higher in periodontitis patients. |
Romano et al. [106] | Cross-sectional study | 15 UP, 15 TP; 15 controls. | Mass spectrometry-based untargeted proteomics. | Cystatin SN higher in healthy patients and in patients under periodontal active treatment. |
Gonçalves et al. [114] | Cross-sectional study | 10 cases, 10 controls | Mass spectrometry-based untargeted proteomics | Periodontitis patients had higher levels of albumin, hemoglobin, and immunoglobulin, and they had a lower abundance of cystatin compared to the control group. |
Salazar et al. [96] | Cross-sectional study | 20 cases, 20 controls. | Mass spectrometry-based untargeted proteomics | alpha-2-macroglobulin, ceruloplasmin, complement C3, alpha-2-HS-glycoprotein, fibrinogen alpha chain higher in periodontitis patients- |
Chaiyarit et al. [115] | Cross-sectional study | 30 cases, 30 controls. | Mass spectrometry-based untargeted proteomics | No identification of specific proteins |
Targeted Analysis | Untargeted Analysis |
---|---|
Hypothesis-driven | Hypothesis-generating |
Subset analysis | Global/Comprehensive analysis |
Correlated to reference standards | Correlated to the database/libraries |
Identification already know | Qualitative identification |
Absolute quantification | Relative quantification |
Feature | NMR Spectroscopy | Mass Spectrometry (MS) | References |
---|---|---|---|
Sensitivity | Low (µM range), although improving with hyperpolarization techniques. | Very high (nM–pM range), especially with HR-MS. | [125,128,129,130,131] |
Quantification | Highly accurate, can quantify multiple analytes. | Quantification is relative unless internal standards are used | [125,128,129,130,131] |
Reproducibility | High, robust across time and between labs. | Moderate; targeted methods are reproducible, and untargeted methods need strict quality control. | [125,132,133] |
Data Complexity | Moderate; relatively easier to interpret. | High; requires extensive data processing and normalization. | [125] |
Throughput | Moderate (10–20 samples/day); limited by long acquisition times in 2D. | High (50–200+ samples/day), depending on the platform. | [125] |
Method Development | Stable protocols; recent improvements in 2D NMR and hyperpolarization. | Rapidly evolving, flexible with multiple ionization and chromatographic modes. | [125] |
Limitations | Low sensitivity–Long acquisition times for complex spectra. | - Ion suppression - Requires strict QC - Less robust than NMR | [125,128,129,130,131] |
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Amato, M.; Polizzi, A.; Blasi, A.; Grippaudo, C.; Isola, G. Untargeted Salivary Metabolomics and Proteomics: Paving the Way for Early Detection of Periodontitis. Appl. Sci. 2025, 15, 6642. https://doi.org/10.3390/app15126642
Amato M, Polizzi A, Blasi A, Grippaudo C, Isola G. Untargeted Salivary Metabolomics and Proteomics: Paving the Way for Early Detection of Periodontitis. Applied Sciences. 2025; 15(12):6642. https://doi.org/10.3390/app15126642
Chicago/Turabian StyleAmato, Mariacristina, Alessandro Polizzi, Andrea Blasi, Cristina Grippaudo, and Gaetano Isola. 2025. "Untargeted Salivary Metabolomics and Proteomics: Paving the Way for Early Detection of Periodontitis" Applied Sciences 15, no. 12: 6642. https://doi.org/10.3390/app15126642
APA StyleAmato, M., Polizzi, A., Blasi, A., Grippaudo, C., & Isola, G. (2025). Untargeted Salivary Metabolomics and Proteomics: Paving the Way for Early Detection of Periodontitis. Applied Sciences, 15(12), 6642. https://doi.org/10.3390/app15126642