## 7 Targeted and Untargeted Approaches in Biomarker Discovery  
Early diagnosis of periodontitis through a rapid, accurate, and non-invasive method is highly desirable to minimize the individual and epidemiological impact of this widely prevalent disease. Targeted and untargeted salivary metabolomics and proteomics analyses are potential valid approaches that may enable clinicians to get to an early diagnosis of periodontitis. Untargeted analysis aims to detect and compare as many signals as possible related to unknown metabolites in a set of samples. Each metabolite and each protein is identified by referencing a metabolic and proteomic database. This approach can be divided into two categories: fingerprinting, which focuses on analyzing intracellular metabolites and proteins, and footprinting, which examines metabolites and proteins found in the surrounding environment or culture medium. Both methods involve rapid analysis and typically do not include precise quantification of the metabolites [116]. Targeted analysis focuses on selected groups of metabolites and proteins, aiming to detect and accurately measure their concentrations in biological samples [117; 118]. This method is often employed for metabolomic and proteomic profiling when the aim is to examine a limited set of metabolites and proteins involved in specific biological pathways. As previously mentioned, untargeted approaches are better than targeted approaches because they involve a complete analysis of the whole composition of saliva, without a prior selection. This allows for the identification of salivary components as biomarkers that would have been discarded if a targeted analysis had been conducted (Table 6) [119]. \begin{table}
\begin{tabular}{c c} \hline \hline
**Targeted Analysis** & **Untargeted Analysis** \\ \hline 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 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Differences between targeted and untargeted analysis. Target studies focus research on several known metabolites, while untargeted studies allow for a more comprehensive evaluation of metabolomic and proteomic profiles [119]. Using untargeted analysis methods on salivary samples to reveal novel biomarkers for early detection of periodontitis is advantageous. In fact, in the early stages of periodontitis, changes in known biomarkers may be subtle or absent. Untargeted approaches can uncover novel, low-abundance molecules or complex biomarker signatures that targeted approaches would miss [120,121]. In addition, due to the multifactorial nature of periodontitis, involving host immune responses, microbial dysbiosis, and inflammatory signaling, untargeted approaches can profile the full molecular spectrum, offering a comprehensive view of pathogenesis. Untargeted analyses are not limited to preselected markers; thus, they reduce the risk of overlooking important but unknown indicators of early disease [121]. Finally, even though untargeted approaches are very advantageous, there is still the need to combine them with targeted approaches. Compounds found by untargeted analysis needs to be confirmed and validated; thus, targeted analysis provides a precise quantification of specific biomarkers identified during the exploratory phase, conducted with an untargeted approach [122,123]. It offers sensitivity, specificity, and quantitative accuracy, which are essential to confirm and validate the results of untargeted analysis [124]. For example, in the study of Bostanci et al. [107], the research of biomarkers associated with periodontitis was conducted at first with a non-targeted shotgun proteomics combined with a label-free quantitative approach. Among the found proteins, only 7 were represented by more than one peptide; therefore, they were included in further comparative quantification.