Search Results (45)

Background: Human proteins exist in numerous modifications—proteoforms—which are promising targets for biomarker studies. In this study, we aimed to generate comparative proteomics data, including proteoform patterns, from hepatocellular carcinoma (HCC) and nonmalignant liver tissues. Methods: To investigate protein profiles and proteoform patterns, we employed a panoramic, integrative top-down proteomics approach: two-dimensional gel electrophoresis (2DE) coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS). Results: We visualized over 2500 proteoform patterns per sample type, enabling the identification of distinct protein signatures and common patterns differentiating nonmalignant and malignant liver cells. Among these, 1270 protein patterns were uniformly observed across all samples. Additionally, 38 proteins—including pyruvate kinase PKM (KPYM), annexin A2 (ANXA2), and others—exhibited pronounced differences in proteoform patterns between nonmalignant and malignant tissues. Conclusions: Most proteoform patterns of the same protein were highly similar, with the dominant peak corresponding to theoretical (unmodified) protein parameters. However, certain proteins displayed altered proteoform patterns and additional proteoforms in cancer compared to controls. These proteins were prioritized for further characterization. Full article

Colorectal cancer (CRC) and lung cancer (LC) are leading causes of cancer-related mortality, highlighting the need for minimally invasive diagnostic, prognostic, and predictive markers for these cancers. Proteins secreted by a tumor into the extracellular space directly, known as the tumor secretome, as well as proteins in the extra-cellular vesicles (EVs), represent an attractive source of biomarkers for CRC and LC. We performed proteomic analyses on secretome and EV samples from LC (A549, NCI-H23, NCI-H460) and CRC (Caco2, HCT116, HT-29) cell lines and targeted mass spectrometry on EVs from plasma samples of 20 patients with CRC and 19 healthy controls. A total of 782 proteins were identified across the CRC and LC secretome and EV samples. Of these, 22 and 44 protein markers were significantly elevated in the CRC and LC samples, respectively. Functional annotation revealed enrichment in proteins linked to metastasis and tumor progression for both cancer types. In EVs isolated from the plasma of patients with CRC, ITGB3, HSPA8, TUBA4A, and TLN1 were reduced, whereas FN1, SERPINA1, and CST3 were elevated, compared to healthy controls. These findings support the development of minimally invasive liquid biopsy methods for the detection, prognosis, and treatment monitoring of LC and CRC. Full article

Background/Objectives. Damage of the gastrointestinal mucosa is a major side effect of the anticancer drug 5-fluorouracil (5-FU). Insight into the molecular pathogenesis of 5-FU-induced gut mucositis is expected to justify the strategies of prophylaxis. Methods. We analyzed intestinal specimens obtained from Balb/c mice treated with 70 mg/kg 5-FU daily for up to 6 days. Results. Manifestations of mucositis in the ileum and the colon included diarrhea, weight loss, and morphological lesions. The proteomic analysis revealed dozens of differentially expressed proteins governed by a set of master regulator proteins that regulated downstream pathways culminating in the complexes of specific transcription factors. Among the most important mechanisms of 5-FU-induced gut damage predicted by bioinformatics tools was stimulation of insulin-like growth factor 1 concomitant with inhibition of insulin receptor substrate 1, suggesting an involvement of the insulin pathway. Furthermore, the levels of 14-3-3γ protein and epinephrin B2 tyrosine kinase were interpreted as key inhibitory effects of 5-FU. These changes were detectable in the ileum as well as in the colon, pointing to the commonality of 5-FU responses across the gut. Conclusion. These results demonstrated a hierarchical network of gut injury mechanisms differentially regulated in the course of the emergence of 5-FU-induced mucositis. Full article

Limit of detection (LoD) is a term that is used to characterize the sensitivity of an analytical method. The existing limitation of the sensitivity of analysis using modern mass spectrometry methods has been experimentally shown to be a limiting factor in the application of proteomic technologies in medicine. This article proposes a concept of a new technology that will set a new vector of development in the development of systems for solving problems of medical diagnostics and deals with theoretical and practical aspects of creating a new technology for the detection of single biomacromolecules (in particular, proteins) in biological samples. Such technology should be based on the principle of signal registration similar to that used in a Geiger counter (also known as a Geiger–Müller counter or G-M counter), a device that automatically counts the number of ionizing particles that hit it. This counter is free from probabilistic components; it registers a signal if there is at least one target molecule in the analysis chamber. Predictive medical diagnostics require technology based on methods where sensitivity allows for the detection of single marker molecules in a biological sample volume of 1–10 µL, the smallest volume of biomaterial used in laboratory diagnostics. Creation of a detector with a sensitivity of 10⁻¹⁸ M would allow for the detection of one molecule in 1 µL of the sample, which fundamentally makes this approach analogous to a G-M counter for solutions. To date, bioanalytical methods are limited to a sensitivity of 10⁻¹² M (which is approximately 1 million molecules per 1 μL), which is insufficient to capture the early stages of pathological processes. Full article

Experience shows that dilution of lubricating oil with diesel oil is unfavorable to the engine, causing issues including deterioration of engine performance, shortening of oil life, and reduction in engine reliability and safety. This paper presents the verification of the hypothesis that the changes in lubricity, friction coefficient, and decreasing oil film thickness (using a relative approach, given as a percentage) are similar for lubricating oil and diesel mixtures prepared from fresh lubricating oil and used lubricating oil. To validate this hypothesis, an experiment is conducted using a high-frequency reciprocating rig (HFFR), in which the lubricity is determined by the corrected average wear scar WS_1.4, the coefficient of friction μ, and the percentage relative decrease in oil film thickness r. A qualitative visual assessment of the wear scars on the test specimens is also performed after the HFFR tests. The testing covers mixtures of SAE 30 grade Marinol CB-30 RG1230 lubricating oil with Orlen Efecta Diesel Biodiesel. The used lubricating oil is extracted from the circulating lubrication system of a supercharged, trunk-piston, four-stroke ZUT Zgoda Sulzer 5 BAH 22 engine installed in the laboratory of ship power plants of the Maritime University of Szczecin. Mixtures for the experiment are prepared for fresh lubricating oil with diesel oil and used lubricating oil with diesel oil. Mixtures of these lubricating oils with diesel oil are examined for diesel oil concentrations in the mixture equal to 1, 2, 5, 10, 15, and 20% m/m. The results of the experiment confirm the hypothesis, proving that, for up to 20% m/m diesel oil concentration in lubricating oil, the changes in the lubricity of used lubricating oil diluted with diesel oil can be evaluated based on reference data prepared for mixtures of diesel oil with fresh lubricating oil. The linear approximation of μ and r trends is made with a certain margin of error we estimated. The experiment also confirms the results of previous studies which state that oil aging products in small quantities contribute to improved lubricity. Full article

This article presents the verification of the hypothesis on using certain approximation curves in the evaluation of used lubricating oil. These curves are plotted for fresh lubricating oil to approximate the parameters of lubricating oil diluted with diesel oil. To confirm the hypothesis, an experiment is conducted to determine the flash point, initial boiling point, density at 15 °C, kinematic viscosity at 40 °C and 100 °C, and viscosity index. The analysis covers fresh oil and used SAE 30 grade Marinol CB-30 RG1230 oil taken from the circulating lubrication system of a supercharged, trunk-piston, 4-stroke ZUT Zgoda Sulzer 5 BAH 22 engine that is located in the Marine Power Plant Laboratory of the Maritime University of Szczecin. Undiluted lubricating oils (both fresh and used) and mixtures of lubricating oils with diesel oil are examined for diesel oil concentrations in the mixture equal to 1, 2, 5, 10, 15, and 20% m/m. Orlen Efecta Diesel Biodiesel is used to prepare the mixtures. The functions approximating the parameters for fresh oil are determined and adapted to describe the variation of the same parameters for the used lubricating oil. For each case, the coefficient of determination, the maximum relative error of the model fitting to the experimental results, and the root mean square error (RMSE) are determined. In the experiment, the variation in the parameters of the used lubricating oil remained the same as for fresh oil parameters. Thus, the research hypothesis is confirmed. Full article

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3–72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML). Full article

This work demonstrates the use of a modified mica to concentrate proteins, which is required for proteomic profiling of blood plasma by mass spectrometry (MS). The surface of mica substrates, which are routinely used in atomic force microscopy (AFM), was modified with a photocrosslinker to allow “irreversible” binding of proteins via covalent bond formation. This modified substrate was called the AFM chip. This study aimed to determine the role of the surface and crosslinker in the efficient concentration of various types of proteins in plasma over a wide concentration range. The substrate surface was modified with a 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) photocrosslinker, activated by UV irradiation. AFM chips were incubated with plasma samples from a healthy volunteer at various dilution ratios (10²X, 10⁴X, and 10⁶X). Control experiments were performed without UV irradiation to evaluate the contribution of physical protein adsorption to the concentration efficiency. AFM imaging confirmed the presence of protein layers on the chip surface after incubation with the samples. MS analysis of different samples indicated that the proteomic profile of the AFM-visualized layers contained common and unique proteins. In the working series of experiments, 228 proteins were identified on the chip surface for all samples, and 21 proteins were not identified in the control series. In the control series, a total of 220 proteins were identified on the chip surface, seven of which were not found in the working series. In plasma samples at various dilution ratios, a total of 146 proteins were identified without the concentration step, while 17 proteins were not detected in the series using AFM chips. The introduction of a concentration step using AFM chips allowed us to identify more proteins than in plasma samples without this step. We found that AFM chips with a modified surface facilitate the efficient concentration of proteins owing to the adsorption factor and the formation of covalent bonds between the proteins and the chip surface. The results of our study can be applied in the development of highly sensitive analytical systems for determining the complete composition of the plasma proteome. Full article

Problems with the male reproductive system are of both medical and social significance. As a rule, spermatozoa and seminal plasma proteomes are investigated separately to assess sperm quality. The current study aimed to compare ejaculate proteomes with spermatozoa and seminal plasma protein profiles regarding the identification of proteins related to fertility scores. A total of 1779, 715, and 2163 proteins were identified in the ejaculate, seminal plasma, and spermatozoa, respectively. Among these datasets, 472 proteins were shared. GO enrichment analysis of the common proteins enabled us to distinguish biological processes such as single fertilization (GO:0007338), spermatid development (GO:0007286), and cell motility (GO:0048870). Among the abundant terms for GO cellular components, zona pellucida receptor complex, sperm fibrous sheath, and outer dense fiber were revealed. Overall, we identified 139 testis-specific proteins. For these proteins, PPI networks that are common in ejaculate, spermatozoa, and seminal plasma were related to the following GO biological processes: cilium movement (GO:0003341), microtubule-based movement (GO:0007018), and sperm motility (GO:0097722). For ejaculate and spermatozoa, they shared 15 common testis-specific proteins with spermatogenesis (GO:0007283) and male gamete generation (GO:0048232). Therefore, we speculated that ejaculate-based proteomics could yield new insights into the peculiar reproductive physiology and spermatozoa function of men and potentially serve as an explanation for male infertility screening. Full article

Over the last few years, a large number of studies have been conducted on the monitoring of human behavior remaining beyond conscious control. One area of application for such monitoring systems is lie detection. The most popular method currently used for this purpose is polygraph examination, which has proven its usefulness in the field and in laboratories, but it is not without its drawbacks. Technological advances in data acquisition and automated analysis have ensured that contactless tools are in high demand in security fields like airport screening or pre-employment procedures. As a result, there has been a shift in interest away from traditional polygraph examinations toward the analysis of facial expressions, voice, and speech patterns, as well as eye-tracking signals to detect deceptive behavior. In this paper, we focus on the last aspect, offer a comprehensive overview of two distinct lie detection methodologies based on eye tracking, and examine the commonly used oculomotor feature analysis. Furthermore, we explore current research directions and their results within the context of their potential applications in the field of forensics. We also highlight future research prospects, suggesting the utilization of eye tracking and scan path interpretation methodologies as a potential fully functional alternative for the conventional polygraph in the future. These considerations refer to legal and ethical issues related to the use of new technology to detect lies. Full article

Despite the undisputable role of the protein corona in the biointeractions of liposome drug carriers, the field suffers from a lack of knowledge regarding the patterns of protein deposition on lipid surfaces with different compositions. Here, we investigated the protein coronas formed on liposomes of basic compositions containing combinations of egg phosphatidylcholine (PC), palmitoyloleoyl phosphatidylglycerol (POPG), and cholesterol. Liposome−protein complexes isolated by size-exclusion chromatography were delipidated and analyzed using label-free LC-MS/MS. The addition of the anionic lipid and cholesterol both affected the relative protein abundances (and not the total bound proteins) in the coronas. Highly anionic liposomes, namely those containing 40% POPG, carried corona enriched with cationic proteins (apolipoprotein C1, beta-2-glycoprotein 1, and cathelicidins) and were the least stable in the calcein release assay. Cholesterol improved the liposome stability in the plasma. However, the differences in the corona compositions had little effect on the liposome uptake by endothelial (EA.hy926) and phagocytic cells in the culture (U937) or ex vivo (blood-derived monocytes and neutrophils). The findings emphasize that the effect of protein corona on the performance of the liposomes as drug carriers occurs through compromising particle stability rather than interfering with cellular uptake. Full article

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. Full article

Mass spectrometry (MS) is one of the main techniques for protein identification. Herein, MS has been employed for the identification of bovine serum albumin (BSA), which was covalently immobilized on the surface of a mica chip intended for investigation by atomic force microscopy (AFM). For the immobilization, two different types of crosslinkers have been used: 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) and dithiobis(succinimidyl propionate) (DSP). According to the data obtained by using an AFM-based molecular detector, the SuccBB crosslinker was more efficient in BSA immobilization than the DSP. The type of crosslinker used for protein capturing has been found to affect the results of MS identification. The results obtained herein can be applied in the development of novel systems intended for the highly sensitive analysis of proteins with molecular detectors. Full article

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein–protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321. Full article

Vestibular schwannomas are relatively rare intracranial tumors compared to other brain tumors. Data on the molecular features, especially on schwannoma proteome, are scarce. The 41 cerebrospinal fluid (liquor) samples were obtained during the surgical removal of vestibular schwannoma. Obtained peptide samples were analyzed by shotgun LC-MS/MS high-resolution mass spectrometry. The same peptide samples were spiked with 148 stable isotopically labeled peptide standards (SIS) followed by alkaline fractionation and scheduled multiple reaction monitoring (MRM) for quantitative analysis. The natural counterparts of SIS peptides were mapped onto 111 proteins that were Food and Drug Administration (FDA)-approved for diagnostic use. As a result, 525 proteins were identified by shotgun LC-MS/MS with high confidence (at least two peptides per protein, FDR < 1%) in liquor samples. Absolute quantitative concentrations were obtained for 54 FDA-approved proteins detected in at least five experimental samples. Since there is lack of data on the molecular landscape of vestibular schwannoma, the obtained datasets are unique and one of the first in its field. Full article