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.

C‐Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a β chain. Upon ligand binding, C‐Met transmits intracellular signals by a unique multi‐substrate docking site. C‐Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C‐Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C‐Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure. Epitope peptides of aptamer‐C‐Met complexes were identified by proteolytic affinity‐mass spectrometry in combination with SPR biosensor analysis (PROTEX‐SPR‐MS), using high‐pressure proteolysis for efficient digestion. High affinities (KD, 80–510 nM) were determined for aptamer‐C‐Met complexes, with two‐step binding suggested by kinetic analysis. A linear epitope, C‐Met (381–393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C‐Met (524–543) and C‐Met (557–568). Structure modeling of C‐Met‐aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C‐Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.

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.

In this study, the nature of lentil protein-tannic acid (LPTA) interaction and its effect on in vitro pepsin digestion were investigated. LPTA mixtures containing 1% w/v LP and 0.001–0.5% TA were prepared and characterized in terms of particle size, thermal properties, and secondary and tertiary structures. A 20-fold increase in particle size was observed in LPTA0.5% compared to LP control (without TA), indicating aggregation. Static quenching of tryptophan residues within the protein hydrophobic folds was observed. Increasing TA levels also enhanced protein thermal stability. Over 50% reduction in free amino groups of LPTA 0.5%, relative to LP, was observed after pepsin digestion. Cleavage specificity of pepsin and peptidomic profile of LP were modified by the presence of TA in LPTA 0.5%. This study showed that 0.5% w/v TA induced protein aggregation and reduced LP digestibility by hindering the accessibility of pepsin to the protein network, thus modifying the profile of released peptides.