Background/Objectives: Tarin exhibits immunomodulatory and antiproliferative properties against several tumor cell lines. Nano-encapsulation in liposomes enhances its therapeutic potential by improving protein stability, bioavailability, and sustained release. Previous studies demonstrated that nano-encapsulated tarin induces cell cycle arrest, migration inhibition, apoptosis, and autophagy in triple-negative breast cancer cells; however, the molecular mechanisms underlying these effects remain poorly understood. To investigate the proteomic response elicited by nano-encapsulated tarin, MDA-MB-231 cells were treated for 24 and 48 h. Methods: Intracellular proteins were extracted, digested with trypsin, and analyzed by label-free LC-2D-MS/MS using HDMSE acquisition. Differentially expressed proteins were identified and quantified using the Progenesis QI platform, and then functional classification and pathway enrichment analyses were performed. Results: A total of 2818 proteins were identified, of which 2150 displayed time-dependent modulation following treatment. After 24 h, cells exhibited an adaptive stress response profile characterized by increased DNA repair proteins (CHEK1, CDK12), migration/remodeling factors (LAMA4, CTTN, A2M), and immune/cell cycle regulators (PER2, HLA-B), while antioxidant proteins (SOD1, GPX1) and BRCA1 were reduced, indicating oxidative stress and DNA damage. After 48 h, the proteomic profile shifted toward cell death, with increased PARK7, OPA1, ATL3, and CASP8 expression, disruption of DNA repair and cell cycle regulators (CHEK1, CDK12, MSH6, KIF2C), and decreased migration-related proteins (LAMA4, CTTN, ITGB3, A2M). Conclusions: Nano-encapsulated tarin promotes a time-dependent transition from early adaptive stress responses to apoptosis, autophagy, cell cycle disruption, and loss of migratory capacity. These findings provide novel insights into the molecular mechanisms underlying tarin antitumoral activity and support its potential as a promising therapeutic strategy against triple-negative breast cancer.

Reliable quantitative proteomic profiling is essential for understanding drug-induced molecular responses in cancer cells. Efficient extraction and digestion of proteins, especially membrane-associated proteins, remain major analytical challenges. Herein, we validated a surfactant-assisted protein extraction and digestion workflow for comprehensive proteome profiling of MDA-MB-231 cell, to establish a baseline reference for drug-response studies at different time points (0, 24, and 48 h). Total proteins were extracted using acid-labile surfactant - RapiGest™ SF - and trypsinized, followed by two-dimensional liquid chromatography coupled to high-definition data-independent mass spectrometry (2D-LC-HDMSE). Data are available via ProteomeXchange with identifier PXD076864. Label-free quantitative analysis identified 2,818 proteins, achieving performance comparable or superior to urea-based and filter-aided sample preparation (FASP) protocols under similar chromatographic conditions. The employed workflow was able to discriminate distinct cellular stages associated with culture establishment and progression over 48h. This temporal proteomic profiling enabled the mapping of protein expression modulated during adhesion, cytoskeletal reorganization, metabolic adaptation, and high-density growth. Data indicate that at high-density culture, intrinsic pathways for immunogenicity are downregulated, a typical evading mechanism of tumor masses and metastatic niches. This study provides a high-depth, reproducible, and MS-compatible strategy to characterize MDA-MB-231 baseline proteome, which may be a reference for drug-response studies.