The proteins of extracellular vesicles (EVs) that originate from tumors reflect the producer cells’ proteomes and can be detected in biological fluids. Thus, EVs provide proteomic signatures that are of great interest for screening and predictive cancer diagnostics. By applying targeted mass spectrometry with stable isotope-labeled peptide standards, we assessed the levels of 28 EV-associated proteins, including the conventional exosome markers CD9, CD63, CD81, CD82, and HSPA8, in vesicles derived from the lung cancer cell lines NCI-H23 and A549. Furthermore, we evaluated the detectability of these proteins and their abundance in plasma samples from 34 lung cancer patients and 23 healthy volunteers. The abundance of TLN1, TUBA4A, HSPA8, ITGB3, TSG101, and PACSIN2 in the plasma of lung cancer patients was measured using targeted mass spectrometry and compared to that in plasma from healthy volunteers. The most diagnostically potent markers were TLN1 (AUC, 0.95), TUBA4A (AUC, 0.91), and HSPA8 (AUC, 0.88). The obtained EV proteomic signature allowed us to distinguish between the lung adenocarcinoma and squamous cell carcinoma histological types. The proteomic cargo of the extracellular vesicles represents a promising source of potential biomarkers.

Virtual and experimental 2DE coupled with ESI LC‐MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI‐TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC‐MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw (http://web.expasy.org/compute_pi/pi_tool‐doc.html). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.

Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and pharmacological activities. At doses of 100 mg/kg and above, isatin is neuroprotective in different experimental models of neurodegeneration. Good evidence exists that its effects are realized via interaction with numerous isatin-binding proteins identified in the brain and peripheral tissues studied. In this study, we investigated the effect of a single dose administration of isatin to mice (100 mg/kg, 24 h) on differentially expressed proteins and a profile of the isatin-binding proteins in brain hemispheres. Isatin administration to mice caused downregulation of 31 proteins. However, these changes cannot be attributed to altered expression of corresponding genes. Although at this time point isatin influenced the expression of more than 850 genes in brain hemispheres (including 433 upregulated and 418 downregulated genes), none of them could account for the changes in the differentially expressed proteins. Comparative proteomic analysis of brain isatin-binding proteins of control and isatin-treated mice revealed representative groups of proteins sensitive to isatin administration. Control-specific proteins (n = 55) represent specific targets that interact directly with isatin. Appearance of brain isatin-binding proteins specific to isatin-treated mice (n = 94) may be attributed to the formation of new clusters of protein–protein interactions and/or novel binding sites induced by a high concentration of this regulator (ligand-induced binding sites). Thus, isatin administration produces multiple effects in the brain, which include changes in gene expression and also profiles of isatin-binding proteins and their interactomes. Further studies are needed for deeper insight into the mechanisms of the multilevel changes in the brain proteome induced by isatin. In the context of the neuroprotective action, these changes may be aimed at interruption of pathological links that begin to form after initiation of pathological processes.

Tumor-derived extracellular vesicles (EVs), including exosomes, contain proteins that mirror the molecular landscape of producer cells. Being potentially detectible in biological fluids, EVs are of great interest for the screening of cancer biomarkers. To reveal universal, tissue-specific, and line-specific markers, we performed label-free mass spectrometric profiling of EVs originating from the human colon cancer cell lines Caco-2, HT29, and HCT-116, as well as from the lung cancer cell lines NCI-H23 and A549. A total of 651 proteins was identified in the EV samples using at least two peptides. These proteins were highly enriched in exosome markers. We found 11 universal, eight tissue-specific, and 29 line-specific markers, the levels of which were increased in EVs compared to the whole lysates. The EV proteins were involved in the EGFR, Rap1, integrin, and microRNA signaling associated with metastasis and cancer progression. An EV protein-based assay could be developed as a liquid biopsy tool.

The proteins of extracellular vesicles (EVs) provide proteomic signatures that reflect molecular features of EV-producing cells, including cancer cells. Detection of cancer cell EV proteins is of great interest due to the development of novel predictive diagnostic approaches. Using targeted mass spectrometry with stable-isotope-labeled peptide standards (SIS), we measured in this study the levels of 34 EV-associated proteins in vesicles and whole lysate derived from the colorectal cancer (CRC) cell lines Caco-2, HT29 and HCT116. We also evaluated the abundance of 13 EV-associated proteins (FN1, TLN1, ITGB3, HSPA8, TUBA4A, CD9, CD63, HSPG2, ITGB1, GNAI2, TSG101, PACSIN2, and CDC42) in EVs isolated from blood plasma samples from 11 CRC patients and 20 healthy volunteers. Downregulation of TLN1, ITGB3, and TUBA4A with simultaneous upregulation of HSPG2 protein were observed in cancer samples compared to healthy controls. The proteomic cargo of the EVs associated with CRC represents a promising source of potential prognostic markers.