Proteomic Stability and Ex Vivo Compatibility of a Processed Phospholipoproteic Secretome-Derived Formulation

Background: Processed extracellular lipid-protein preparations require rigorous analytical characterization to determine whether their compositional profile, processing stability, and short-term cellular compatibility are preserved across storage and handling conditions. Methods: In this study, we quantitatively characterized a processed phospholipoproteic secretome-derived formulation under fresh, concentrated, cryopreserved, and lyophilized conditions. Results: Label-free quantitative proteomic analyses performed using timsTOF Pro mass spectrometry coupled to dia-PASEF acquisition identified 574 human proteins across all experimental conditions following predefined analytical quality criteria. Comparative analyses demonstrated preservation of the overall structural proteomic profile following processing and storage procedures, with retention of membrane-associated and extracellular structural proteins consistently exceeding 90% relative to the fresh reference condition. Quantitative reproducibility remained high across all experimental groups, with coefficients of variation ranging from 3.0% to 4.5% and strong inter-replicate Pearson correlations. Principal component analysis, hierarchical clustering, peptide/protein overlap analyses, and differential expression profiling demonstrated limited proteomic divergence while preserving the majority of quantified proteins within conserved abundance ranges. Complementary real-time live-cell kinetic imaging performed in non-malignant dermal-derived cells using the IncuCyte^® S3 platform demonstrated stable short-term confluence kinetics and cellular viability exceeding 92% over 48 h across all evaluated formulations. No sustained proliferative suppression or detectable morphological evidence of cytotoxicity was observed. Collectively, these findings support the preservation of compositional stability, analytical reproducibility, and short-term ex vivo cellular compatibility across defined processing and storage conditions. These integrated proteomic and kinetic datasets provide a quantitative framework for the analytical evaluation of processed extracellular phospholipoproteic preparations, while functional barrier activity, membrane incorporation, lipid raft engagement, and long-term tissue-level effects remain to be addressed in dedicated future studies.