Small membrane-derived extracellular vesicles have been proposed as participating in several cancer diseases, including breast cancer (BC). We performed a phosphoproteomic analysis of breast cancer-derived small extracellular vesicles (sEVs) to provide insight into the molecular and cellular regulatory mechanisms important for breast cancer tumor progression and metastasis. We examined three cell line models for breast cancer: MCF10A (non-malignant), MCF7 (estrogen and progesterone receptor-positive, metastatic), and MDA-MB-231 (triple-negative, highly metastatic). To obtain a comprehensive overview of the sEV phosphoproteome derived from each cell line, effective phosphopeptide enrichment techniques IMAC and TiO₂, followed by LC-MS/MS, were performed. The phosphoproteome was profiled to a depth of 2003 phosphopeptides, of which 207, 854, and 1335 were identified in MCF10A, MCF7, and MDA-MB-231 cell lines, respectively. Furthermore, 2450 phosphorylation sites were mapped to 855 distinct proteins, covering a wide range of functions. The identified proteins are associated with several diseases, mostly related to cancer. Among the phosphoproteins, we validated four enzymes associated with cancer and present only in sEVs isolated from MCF7 and MDA-MB-231 cell lines: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6). With the exception of PFKM, the specific activity of these enzymes was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study demonstrates that sEVs contain functional metabolic enzymes that could be further explored for their potential use in early BC diagnostic and therapeutic applications.

Breast cancer (BC) is the second most frequently diagnosed cancer and accounts for approximately 25% of new cancer cases in Canadian women. Using biomarkers as a less-invasive BC diagnostic method is currently under investigation but is not ready for practical application in clinical settings. During the last decade, extracellular vesicles (EVs) have emerged as a promising source of biomarkers because they contain cancer-derived proteins, RNAs, and metabolites. In this study, EV proteins from small EVs (sEVs) and medium EVs (mEVs) were isolated from BC MDA-MB-231 and MCF7 and non-cancerous breast epithelial MCF10A cell lines and then analyzed by two approaches: global proteomic analysis and enrichment of EV surface proteins by Sulfo-NHS-SS-Biotin labeling. From the first approach, proteomic profiling identified 2459 proteins, which were subjected to comparative analysis and correlation network analysis. Twelve potential biomarker proteins were identified based on cell line-specific expression and filtered by their predicted co-localization with known EV marker proteins, CD63, CD9, and CD81. This approach resulted in the identification of 11 proteins, four of which were further investigated by Western blot analysis. The presence of transmembrane serine protease matriptase (ST14), claudin-3 (CLDN3), and integrin alpha-7 (ITGA7) in each cell line was validated by Western blot, revealing that ST14 and CLDN3 may be further explored as potential EV biomarkers for BC. The surface labeling approach enriched proteins that were not identified using the first approach. Ten potential BC biomarkers (Glutathione S-transferase P1 (GSTP1), Elongation factor 2 (EEF2), DEAD/H box RNA helicase (DDX10), progesterone receptor (PGR), Ras-related C3 botulinum toxin substrate 2 (RAC2), Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), Aconitase 2 (ACO2), UTP20 small subunit processome component (UTP20), NEDD4 binding protein 2 (N4BP2), Programmed cell death 6 (PDCD6)) were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. In total, 846 surface proteins were identified from the second approach, of which 11 were already known as BC markers. This study supports the proposition that Evs are a rich source of known and novel biomarkers that may be used for non-invasive detection of BC. Furthermore, the presented datasets could be further explored for the identification of potential biomarkers in BC.

Cancer-derived small extracellular vesicles have been proposed as promising potential biomarkers for diagnosis and prognosis of breast cancer (BC). We performed a proteomic study of lysine acetylation of breast cancer-derived small extracellular vesicles (sEVs) to understand the potential role of the aberrant acetylated proteins in the biology of invasive ductal carcinoma and triple-negative BC. Three cell lines were used as models for this study: MCF10A (non-metastatic), MCF7 (estrogen and progesterone receptor-positive, metastatic) and MDA-MB-231 (triple-negative, highly metastatic). For a comprehensive protein acetylation analysis of the sEVs derived from each cell line, acetylated peptides were enriched using the anti-acetyl-lysine antibody, followed by LC-MS/MS analysis. In total, there were 118 lysine-acetylated peptides, of which 22, 58 and 82 have been identified in MCF10A, MCF7 and MDA-MB-231 cell lines, respectively. These acetylated peptides were mapped to 60 distinct proteins and mainly identified proteins involved in metabolic pathways. Among the acetylated proteins identified in cancer-derived sEVs from MCF7 and MDA-MB-231 cell lines are proteins associated with the glycolysis pathway, annexins and histones. Five acetylated enzymes from the glycolytic pathway, present only in cancer-derived sEVs, were validated. These include aldolase (ALDOA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK1), enolase (ENO) and pyruvate kinase M1/2 (PKM). For three of these enzymes (ALDOA, PGK1 and ENO) the specific enzymatic activity was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study reveals that sEVs contain acetylated glycolytic metabolic enzymes that could be interesting potential candidates for early BC diagnostics.

Membrane-derived extracellular vesicles, referred to as microvesicles (MVs), have been proposed to participate in several cancer diseases. In this study, MV fractions were isolated by differential ultracentrifugation from a metastatic breast cancer (BC) cell line MDA-MB-231 and a non-cancerous breast cell line MCF10A, then analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 1519 MV proteins were identified from both cell lines. The data obtained were compared to previously analyzed proteins from small extracellular vesicles (sEVs), revealing 1272 proteins present in both MVs and sEVs derived from the MDA-MB-231 cell line. Among the 89 proteins unique to MDA-MB-231 MVs, three enzymes: ornithine aminotransferase (OAT), transaldolase (TALDO1) and bleomycin hydrolase (BLMH) were previously proposed as cancer therapy targets. These proteins were enzymatically validated in cells, sEVs, and MVs derived from both cell lines. The specific activity of OAT and TALDO1 was significantly higher in MDA-MB-231-derived MVs than in MCF10A MVs. BLMH was highly expressed in MDA-MB-231-derived MVs, compared to MCF10A MVs. This study shows that MVs carry functional metabolic enzymes and provides a framework for future studies of their biological role in BC and potential in therapeutic applications.

Egg white protein hydrolysate generated with pepsin was investigated for the presence of peptides with self-assembly and hydrogelation properties. Incubation of the hydrolysates for 16 h resulted in aggregates with significantly (p < 0.05) lower free amino nitrogen and sulfhydryl contents, and higher particle diameter and surface hydrophobicity compared to the hydrolysates. LC-MS/MS analysis of the aggregates resulted in identification of 429 ovalbumin-derived peptides, among which the top-six aggregation-prone peptides IFYCPIAIM, NIFYCPIAIM, VLVNAIVFKGL, YCPIAIMSA, MMYQIGLF, and VYSFSLASRL were predicted using AGGRESCAN by analysis of the aggregation “Hot Spots”. NIFYCPIAIM had the highest thioflavin T fluorescence intensity, particle diameter (5611.3 nm), and polydispersity index (1.0) after 24 h, suggesting the formation of β-sheet structures with heterogeneous particle size distribution. Transmission electron microscopy of MMYQIGLF, and VYSFSLASRL demonstrated the most favorable peptide self-assembly, based on the formation of densely packed, intertwined fibrils. Rheological studies confirmed the viscoelastic and mechanical properties of the hydrogels, with IFYCPIAIM, NIFYCPIAIM, VLVNAIVFKGL, and VYSFSLASRL forming elastic solid hydrogels (tan δ < 1), while YCPIAIMSA and MMYQIGLF formed viscous liquid-like hydrogels (tan δ > 1). The results provide valuable insight into the influence of peptide sequence on hydrogelation and self-assembly progression, and prospects of food peptides in biomaterial applications.

Systematic mapping of protein–ligand interactions is essential for understanding biological processes and drug mechanisms. Peptide-centric local stability assay (PELSA) is a powerful tool for detecting these interactions and identifying potential binding sites. However, its original workflow is limited in throughput, sample compatibility and accessible protein targets. Here, we introduce a high-throughput adaptation—HT-PELSA—that increases sample processing efficiency by 100-fold while maintaining high sensitivity and reproducibility. HT-PELSA substantially extends the capabilities of the original method by enabling sensitive protein–ligand profiling in crude cell, tissue and bacterial lysates, allowing the identification of membrane protein targets in diverse biological systems. We demonstrate that HT-PELSA can precisely and accurately determine binding affinities of small molecule inhibitors, sensitively detect direct and allosteric ATP binding sites, and reveal off-target interactions of a marketed kinase inhibitor in heart tissue. By enhancing scalability, reducing costs and enabling system-wide drug screening across a wide range of sample types, HT-PELSA—when combined with next-generation mass spectrometry—may offer a powerful platform poised to accelerate both drug discovery and basic biological research.