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The search for minimally invasive methods for diagnostics of colorectal cancer (CRC) is the most important task for early diagnostics of the disease and subsequent successful treatment. Human plasma represents the main type of biological material used in the clinical practice; however, the complex dynamic range of substances circulating in it complicates determination of CRC protein markers by the mass spectrometric (MS) method. Studying the proteome of extracellular vesicles (EVs) isolated from human plasma represents an attractive approach for the discovery of tissue-secreted CRC markers. We performed shotgun mass spectrometry analysis of EV samples obtained from plasma of CRC patients and healthy volunteers. This MS analysis resulted in identification of 370 proteins (which were registered by at least two peptides). Stable isotope-free relative quantitation identified 55 proteins with altered abundance in EV samples obtained from plasma samples of CRC patients as compared to healthy controls. Among the EV proteins isolated from blood plasma we found components involved in cell adhesion and the VEGFA-VEGFR2 signaling pathway (TLN1, HSPA8, VCL, MYH9, and others), as well as proteins expressed predominantly by gastrointestinal tissues (polymeric immunoglobulin receptor, PIGR). The data obtained using the shotgun proteomic profiling may be added to the panel for targeted MS analysis of EV-associated protein markers, previously developed using CRC cell models.

Detection of low-copy proteins in complex biological samples is one of the most important issues of modern proteomics. The main reason for inefficient detection of low protein concentrations is the insufficient sensitivity of mass spectrometric detectors and the high dynamic range of protein concentrations. In this study we have investigated the possibilities and limitations of a targeted mass spectrometric analysis using the reconstructed system of standard proteins UPS1 (Universal Proteomic Standard 1) as an example. The study has shown that the sensitivity of the method is affected by the concentration of target proteins of the UPS1 system, as well as by a high level of biological noise modelled by proteins of whole E. coli cell lysate. The limitations of the method have been overcome by concentrating and pre-fractionating the sample peptides in a reversed phase chromatographic system under alkaline elution conditions. Proteomic analysis of the biological sample (proteins of the human hepatocellular carcinoma cell line HepG2 encoded by genes of human chromosome 18) showed an increase in the sensitivity of the method as compared to the standard targeted mass spectrometric analysis. This culminated in registration of 94 proteins encoded by genes located on human chromosome18.

Parkinsonism in rats induced by the pesticide rotenone is one of the most adequate models of Parkinson's disease (PD). Isatin (indole-2,3-dione) is an endogenous regulator found in mammals and humans and exhibiting a wide range of biological activities mediated by numerous isatin-binding proteins, including those associated with neurodegenerative pathology. A course of rotenone administration to rats caused behavioral impairments and changes in the profile and relative content of isatin-binding proteins in the brain. In this study, we have investigated the delayed neuroprotective effect of isatin (5 days after completion of the course of rotenone administration) on behavioral reactions and the relative content of isatin-binding proteins in the brain of rats with rotenone-induced experimental parkinsonism. Although during this period the rats retained locomotor dysfunction, the proteomic analysis data (profile of isatin-binding proteins in the brain and changes in their relative content) differed from the results obtained immediately after completion of the course of rotenone administration. Moreover, all isatin-binding proteins with altered relative content changed during this period are associated to varying degrees with neurodegeneration (many with Parkinson's and Alzheimer's diseases).

Renalase (RNLS) is a recently discovered protein that plays an important role in the regulation of blood pressure by acting inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase that oxidizes isomeric forms of β-NAD(P)H. Extracellular renalase lacking its N-terminal peptide and cofactor FAD exerts various protective effects via non-catalytic mechanisms. Certain experimental evidence exists in the literature that the RP220 peptide (a 20-mer peptide corresponding to the amino acid sequence RNLS 220-239) reproduces a number of non-catalytic effects of this protein, acting on receptor proteins of the plasma membrane. The possibility of interaction of this peptide with intracellular proteins has not been studied. Taking into consideration the known role of RNLS as a possible antihypertensive factor, the aim of this study was to perform proteomic profiling of the kidneys of normotensive and hypertensive rats using RP220 as an affinity ligand. Proteomic (semi-quantitative) identification revealed changes in the relative content of about 200 individual proteins in the kidneys of hypertensive rats bound to the affinity sorbent as compared to the kidneys of normotensive animals. Increased binding of SHR renal proteins to RP220 over the normotensive control was found for proteins involved in the development of cardiovascular pathology. Decreased binding of the kidney proteins from hypertensive animals to RP220 was noted for components of the ubiquitin-proteasome system, ribosomes, and cytoskeleton.

Comparative proteomic analysis of kidney tissue from normotensive (WKY) and spontaneously hypertensive (SHR) rats revealed quantitative and qualitative changes in renal proteins. The number of renal proteins specific for WKY rats (blood pressure 110-120 mm Hg) was 13-16. There were 20-24 renal proteins specific for SHR (blood pressure 180 mm Hg and more). The total number of identified renal proteins common for both rat strains included 972-975 proteins. A pairwise comparison of all possible (SHR-WKY) variants identified 8 proteins specific only for normotensive (WKY) animals, and 7 proteins specific only for hypertensive ones (SHR). Taking into consideration their biological roles, the lack of some enzyme proteins in hypertensive rats (for example, biliverdin reductase A) reduces the production of molecules exhibiting antihypertensive properties, while the appearance of others (e.g. betaine-homocysteine S-methyltransferase 2, septin 2, etc.) can be interpreted as a compensatory reaction. Renal proteins with altered relative content (with more than 2.5-fold change) accounted for no more than 5% of all identified proteins. Among the proteins with an increased relative content in hypertensive animals, the largest group consisted of proteins involved in the processes of energy generation and carbohydrate metabolism, as well as antioxidant and protective proteins. In the context of the development of hypertension, the identified relative changes can apparently be considered compensatory. Among the proteins with the most pronounced decrease in the relative content in hypertensive rats, the dramatic reduction in acyl-CoA medium-chain synthetase-3 (ACSM3) appears to make an important contribution to the development of renal pathology in these animals.

Isatin (indoldione-2,3) is an endogenous biological regulator found in the brain, peripheral tissues, and biological fluids of humans and animals. Its biological activity is realized via isatin-binding proteins, many of which were identified during proteomic profiling of the brain of mice and rats. A number of these proteins are related to the development of neurodegenerative diseases. Previously, using a model of experimental Parkinsonism induced by a seven-day course of rotenone injections, we have observed behavioral disturbances, as well as changes in the profile and relative content of brain isatin-binding proteins. In this study, we have investigated behavioral responses and the relative content of brain isatin-binding proteins in rats with rotenone-induced Parkinsonism 5 days after the last administration of this neurotoxin. Despite the elimination of rotenone, animals exhibited motor and coordination impairments. Proteomic profiling of isatin-binding proteins revealed changes in the relative content of 120 proteins (the relative content of 83 proteins increased and that of 37 proteins decreased). Comparison of isatin-binding proteins characterized by the changes in the relative content observed in the brain right after the last injection of rotenone (n=16) and 5 days later (n=11) revealed only two common proteins (glyceraldehyde-3-phosphate dehydrogenase and subunit B of V-type proton ATPase). However, most of these proteins are associated with neurodegeneration, including Parkinson's and Alzheimer's diseases.