Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

The pathogenesis of primary acquired nasolacrimal duct obstruction (PANDO) remains unclear, with several factors implicated, including anatomical structures, hormones, and tear components. This study explored tear proteins to better understand PANDO etiology by comparing protein expression in tears from patients with PANDO and healthy controls. Tear samples were collected from 22 patients with PANDO (mucous and membranous types) and 8 controls using Weck-Cel sponges. Protein analysis was conducted using LC-MS/MS to identify and quantify tear proteins. Female patients with PANDO had higher numbers of differentially expressed proteins (DEPs) compared with males. Certain DEPs associated with inflammatory pathways or the lacrimal duct epithelium, including SERPINB1, SERPINA3, CTSG, SLPI, and EZR, were identified in male patients. Although this is a preliminary study, our results offer insights into the pathogenesis of PANDO, with potential to distinguish between mucous and membranous subtypes. The potential biomarkers identified in this study could enhance early diagnosis and treatment, shedding light on inflammatory and immune processes in PANDO.

Background: Periodontitis is a chronic inflammatory disease characterized by tissue destruction and immune dysregulation. While gingival crevicular fluid (GCF) serves as a non-invasive biomarker source, its molecular distinctions from periodontal tissue remain underexplored. This study conducted a comparative proteomic analysis of GCF and tissue samples from patients with Stage III–IV periodontitis, integrating differential expression, weighted gene co-expression network analysis, and protein–protein interaction networks to delineate compartment-specific molecular profiles and clarify their respective biological roles in periodontal pathophysiology. Methods: Proteomic data were acquired from GCF and periodontal tissue using label-free LC–MS analysis. Differentially expressed proteins (DEPs) were identified using independent samples t-test ( p < 0.05, |fold-change| > 2). WGCNA was performed to construct co-expression modules and identify functionally related protein clusters, followed by GO enrichment and protein–protein interaction (PPI) analyses using STRING and Cytoscape. Hub proteins were determined through CytoHubba according to centrality measures. Comparative analyses were conducted between tissue and GCF to define inflammation- and repair-related modules and to assess potential molecular interconnections between the two sample types. Results: A total of 4, ¹ 04 proteins were identified in periodontal tissue and 1,546 in GCF. Among these, 1,292 DEPs were detected in tissue and 280 in GCF. Periodontal tissue displayed coordinated upregulation of ribosomal proteins and collagen networks alongside mitochondrial components, indicating repair-oriented structural remodeling and metabolic activation. Conversely, GCF exhibited enrichment of neutrophil-derived immune effectors including MPO, ELANE, CTSG, S100A8, and apolipoproteins, representing innate immune activation. Network integration revealed that GCF and tissue maintained largely distinct molecular profiles with limited cross-compartment connectivity. Conclusions: This comparative proteomic analysis demonstrates that periodontal tissue and GCF represent functionally distinct but complementary biological environments in periodontitis. Periodontal tissue exhibits enhanced structural and metabolic processes, whereas GCF predominantly reflects neutrophil-mediated immune responses. These molecular distinctions provide a basis for developing clinical, compartment-specific biomarker candidates that warrant analytical validation, thereby supporting precision-medicine-relevant diagnostic strategies in periodontal research.

Primary acquired nasolacrimal duct obstruction (PANDO) is a common cause of epiphora in adults, yet the biochemical environment within the nasolacrimal duct (NLD) remains poorly understood. This study aimed to characterize the proteomic composition of NLD lavage fluid and identify subtype-specific molecular features distinguishing membranous and mucinous obstruction. Paired tear and NLD lavage fluid (NLD-LF) samples were collected from patients undergoing dacryoendoscopic recanalization, and proteomic profiling was performed using LC–MS/MS. A total of 1345 proteins were identified in NLD-LF and 767 in tear fluid, revealing a distinct NLD-specific proteome. Although the membranous and mucinous subtypes shared broadly similar protein compositions, differentially expressed proteins highlighted divergent biochemical pathways. The membranous subtype showed enrichment of keratinization-related processes involving KRT1, KRT9, and KLK13, suggesting epithelial remodeling and cornification. In contrast, the mucinous subtype exhibited upregulation of proteins involved in lipid metabolism, carboxylic acid biosynthesis, and sulfur compound metabolism, including ALOX15B, LCAT, and GSTM4, indicating metabolic conditions that promote mucin–lipid interactions, glycan sulfation, and redox-dependent mucin cross-linking. These findings provide new insights into the protein composition of NLD lavage fluid and suggest molecular differences between the membranous and mucinous obstruction subtypes.