From Proteomics to the Analysis of Single Protein Molecules.

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.

The Multienzyme Complex Nature of Dehydroepiandrosterone Sulfate Biosynthesis.

Dehydroepiandrosterone (DHEA), a precursor of steroid sex hormones, is synthesized by steroid 17-alpha-hydroxylase/17,20-lyase (CYP17A1) with the participation of microsomal cytochrome b5 (CYB5A) and cytochrome P450 reductase (CPR), followed by sulfation by two cytosolic sulfotransferases, SULT1E1 and SULT2A1, for storage and transport to tissues in which its synthesis is not available. The involvement of CYP17A1 and SULTs in these successive reactions led us to consider the possible interaction of SULTs with DHEA-producing CYP17A1 and its redox partners. Text mining analysis, protein-protein network analysis, and gene co-expression analysis were performed to determine the relationships between SULTs and microsomal CYP isoforms. For the first time, using surface plasmon resonance, we detected interactions between CYP17A1 and SULT2A1 or SULT1E1. SULTs also interacted with CYB5A and CPR. The interaction parameters of SULT2A1/CYP17A1 and SULT2A1/CYB5A complexes seemed to be modulated by 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Affinity purification, combined with mass spectrometry (AP-MS), allowed us to identify a spectrum of SULT1E1 potential protein partners, including CYB5A. We showed that the enzymatic activity of SULTs increased in the presence of only CYP17A1 or CYP17A1 and CYB5A mixture. The structures of CYP17A1/SULT1E1 and CYB5A/SULT1E1 complexes were predicted. Our data provide novel fundamental information about the organization of microsomal CYP-dependent macromolecular complexes.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Proteomic Analysis of Chr 18 Proteins Using 2D Fractionation.

One of the main goals of the Chromosome-Centric Human Proteome Project (C-HPP) is detection of "missing proteins" (PE2-PE4). Using the UPS2 (Universal proteomics standard 2) set as a model to simulate the range of protein concentrations in the cell, we have previously shown that 2D fractionation enables the detection of more than 95% of UPS2 proteins in a complex biological mixture. In this study, we propose a novel experimental workflow for protein detection during the analysis of biological samples. This approach is extremely important in the context of the C-HPP and the neXt-MP50 Challenge, which can be solved by increasing the sensitivity and the coverage of the proteome encoded by a particular human chromosome. In this study, we used 2D fractionation for in-depth analysis of the proteins encoded by human chromosome 18 (Chr 18) in the HepG2 cell line. Use of 2D fractionation increased the sensitivity of the SRM SIS method by 1.3-fold (68 and 88 proteins were identified by 1D fractionation and 2D fractionation, respectively) and the shotgun MS/MS method by 2.5-fold (21 and 53 proteins encoded by Chr 18 were detected by 1D fractionation and 2D fractionation, respectively). The results of all experiments indicate that 111 proteins encoded by human Chr 18 have been identified; this list includes 42% of the Chr 18 protein-coding genes and 67% of the Chr 18 transcriptome species (Illumina RNaseq) in the HepG2 cell line obtained using a single sample. Corresponding mRNAs were not registered for 13 of the detected proteins. The combination of 2D fractionation technology with SRM SIS and shotgun mass spectrometric analysis did not achieve full coverage, i.e., identification of at least one protein product for each of the 265 protein-coding genes of the selected chromosome. To further increase the sensitivity of the method, we plan to use 5-10 crude synthetic peptides for each protein to identify the proteins and select one of the peptides based on the obtained mass spectra for the synthesis of an isotopically labeled standard for subsequent quantitative analysis. Data are available via ProteomeXchange with the identifier PXD019263.

Nonadditivity in human microsomal drug metabolism revealed in a study with coumarin 152, a polyspecific cytochrome P450 substrate.

We closely characterized 7-Dimethylamino-4-trifluromethylcoumarin (Coumarin 152, C152), a substrate metabolized by multiple P450 species, to establish a new fluorogenic probe for the studies of functional integration in the cytochrome P450 ensemble. Scanning fluorescence spectroscopy and LC/MS-MS were used to characterize the products of N-demethylation of C152 and optimize their fluorometric detection. The metabolism of C152 by the individual P450 species was characterized using the microsomes containing cDNA-expressed enzymes. C152 metabolism in human liver microsomes (HLM) was studied in a preparation with quantified content of eleven P450 species. C152 is metabolized by CYP2B6, CYP3A4, CYP3A5, CYP2C19, CYP1A2, CYP2C9, and CYP2C8 listed in the order of decreasing turnover. The affinities exhibited by CYP3A5, CYP2C9, and CYP2C8 were lower than those characteristic to the other enzymes. The presumption of additivity suggests the participation of CYP3A4, CYP2B6, and CYP2C19 to be 84, 8, and 0.2%, respectively. Contrary to this prediction, inhibitory analysis identified CYP2C19 as the principal C152-metabolizing enzyme. We thoroughly characterize C152 for the studies of drug metabolism in HLM and demonstrate the limitations of the proportional projection approach by providing an example, where the involvement of individual P450 species cannot be predicted from their content.

Toward a systems approach to cytochrome P450 ensemble: interactions of CYP2E1 with other P450 species and their impact on CYP1A2.

In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane. Incorporation of CYP2E1 results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol and Chlorzaxozone. The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3-0.4 nmol/mg protein (or ∼50% CYP2E1 in the P450 pool). The incorporated CYP2E1 becomes a fully functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1. Enrichment of HLM with CYP2E1 results in pronounced changes in the metabolism of 7-ethoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2 suggesting an increase in the involvement of the latter in its metabolism. This effect goes together with an augmentation of the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin. Furthermore, probing the interactions of CYP2E1 with model microsomes containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2.

Intraindividual variability of semen quality, proteome, and sncRNA profiles in a healthy cohort of young adults.

BACKGROUND: Within-subject variability of semen parameters and molecular components of ejaculates in young men remains poorly understood. OBJECTIVES: To investigate intraindividual variability (IIV) of semen parameters and molecular markers in repeated ejaculates from young men. MATERIALS AND METHODS: Semen parameters were assessed in samples collected 6-8 days apart from 164 18-19-year old participants of the Russian Children's Study, a prospective cohort. Subsets of paired samples were used for label-free quantitation and targeted mass-spectrometry of proteins in seminal plasma (SP) and seminal extracellular vesicles (EVs), and for small non-coding RNA (sncRNA) profiling in EVs and spermatozoa using RNA-seq. The mean difference between two ejaculates, within-subject variation, intraclass correlation, and concordance correlation were used to assess IIV for all parameters. Low variability with high reproducibility and high reliability was considered if CVw < 15% and ICC > 0.90, respectively. RESULTS: Analytical variability was low for all investigated parameters in technical replicates. IIV was assessed for basic semen parameters and proteins in SPs and EVs: 319 and 777 proteins, respectively, using untargeted analysis; 9 and 10 proteins using targeted quantification. We also described the IIV for sncRNA, including microRNA, piwi-interacting RNA, tRNA, and tRNA-derived small RNA (tsRNA) in EVs (409 sncRNA and 78 tsRNA) and in spermatozoa (265 sncRNA and 15 tsRNA). We identified 22 and 27 non-overlapping proteins in SP and EVs, respectively, and 46 and 9 sncRNA, including 5 and 0 tsRNA in seminal EVs and spermatozoa, respectively, with low variability. The fatty acid synthase (FAS) had the lowest IIV in both media in targeted protein quantification. DISCUSSION: We identified a number of proteins and sncRNA with low variability among 111 proteins, 176 sncRNA, and 12 tsRNA which were previously suggested as biomarkers of male fertility and reproductive outcomes: lactotransferrin, cysteine-rich secretory protein 3, alpha-1-antichymotrypsin, epididymal sperm-binding protein 1, glutathione S-transferase Mu 3, alpha-1-acid glycoprotein 2, serum amyloid P-component, aminopeptidase N, neprilysin, FAS, and miR-10b-3p, miR-122-5p, miR-205-5p, miR-222-3p, miR-34c-5p, miR-509-3-5p, miR-888-5p, miR-892a, miR-363-3p, miR-941, miR-146a-5p, miR-744-5p. CONCLUSION: These molecules have low IIV and may be promising candidate biomarkers of male fertility and reproductive health.

Age-related changes in human skeletal muscle transcriptome and proteome are more affected by chronic inflammation and physical inactivity than primary aging.

Evaluation of the influence of primary and secondary aging on the manifestation of molecular and cellular hallmarks of aging is a challenging and currently unresolved issue. Our study represents the first demonstration of the distinct role of primary aging and chronic inflammation/physical inactivity - the most important drivers of secondary aging, in the regulation of transcriptomic and proteomic profiles in human skeletal muscle. To achieve this purpose, young healthy people (n = 15), young (n = 8) and older (n = 37) patients with knee/hip osteoarthritis, a model to study the effect of long-term inactivity and chronic inflammation on the vastus lateralis muscle, were included in the study. It was revealed that widespread and substantial age-related changes in gene expression in older patients relative to young healthy people (~4000 genes regulating mitochondrial function, proteostasis, cell membrane, secretory and immune response) were related to the long-term physical inactivity and chronic inflammation rather than primary aging. Primary aging contributed mainly to the regulation of genes (~200) encoding nuclear proteins (regulators of DNA repair, RNA processing, and transcription), mitochondrial proteins (genes encoding respiratory enzymes, mitochondrial complex assembly factors, regulators of cristae formation and mitochondrial reactive oxygen species production), as well as regulators of proteostasis. It was found that proteins associated with aging were regulated mainly at the post-transcriptional level. The set of putative primary aging genes and their potential transcriptional regulators can be used as a resource for further targeted studies investigating the role of individual genes and related transcription factors in the emergence of a senescent cell phenotype.

Number of Detected Proteins as the Function of the Sensitivity of Proteomic Technology in Human Liver Cells.

AIMS: The main goal of the Russian part of C-HPP is to detect and functionally annotate missing proteins (PE2-PE4) encoded by human chromosome 18. To achieve this goal, it is necessary to use the most sensitive methods of analysis. BACKGROUND: However, identifying such proteins in a complex biological mixture using mass spectrometry (MS)-based methods is difficult due to the insufficient sensitivity of proteomic analysis methods. A possible solution to the problem is the pre-fractionation of a complex biological sample at the sample preparation stage. OBJECTIVE: This study aims to measure the detection limit of SRM SIS analysis using a standard set of UPS1 proteins and find a way to enhance the sensitivity of the analysis and to, detect proteins encoded by the human chromosome 18 in liver tissue samples, and compare the data with transcriptomic analysis of the same samples. METHODS: Mass spectrometry, data-dependent acquisition, selected reaction monitoring, highperformance liquid chromatography, data-dependent acquisition in combination with pre-fractionation by alkaline reversed-phase chromatography, selected reaction monitoring in combination with prefractionation by alkaline reversed-phase chromatography methods were used in this study. RESULTS: The results revealed that 100% of UPS1 proteins in a mixture could only be identified at a concentration of at least 10-9 М. The decrease in concentration leads to protein losses associated with technology sensitivity, and no UPS1 protein is detected at a concentration of 10-13 М. Therefore, the two-dimensional fractionation of samples was applied to improve sensitivity. The human liver tissue was examined by selected reaction monitoring and shotgun methods of MS analysis using onedimensional and two-dimensional fractionation to identify the proteins encoded by human chromosome 18. A total of 134 proteins were identified. The overlap between proteomic and transcriptomic data in human liver tissue was ~50%. CONCLUSION: The sample concentration technique is well suited for a standard UPS1 system that is not contaminated with a complex biological sample. However, it is not suitable for use with a complex biological protein mixture. Thus, it is necessary to develop more sophisticated fractionation systems for the detection of all low-copy proteins. This weak convergence is due to the low sensitivity of proteomic technology compared to transcriptomic approaches. Also, total mRNA was used to perform RNA-seq analysis, but not all detected mRNA molecules could be translated into proteins. This introduces additional uncertainty in the data; in the future, we plan to study only translated mRNA molecules-the translatome. Data is available via ProteomeXchange with identifier PXD026997.

Deep proteomic dataset of human liver samples obtained by two-dimensional sample fractionation coupled with tandem mass spectrometry.

The data was acquired from 3 normal human liver tissues by LC-MS methods. The tissue liver samples from male subjects post mortem were obtained from ILSBio LLC (https://bioivt.com/). Liver tissue was frozen in liquid nitrogen, transported and shipped on dry ice. The proteins were extracted and purified followed up by trypsin hydrolysis. The peptide mixture was aliquoted and analyzed by different LC-MS approaches: one-dimensional shotgun LC-MS, two-dimensional LC-MS, two-dimensional SRM SIS (Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards). The Shotgun assay resulted in a qualitative in-depth human liver proteome, and a semi-quantitative iBAQ (intensity-based absolute quantification) value was calculated to show the relative protein content of the sample. Absolute quantitative concentrations of proteins encoded by human chromosome 18 using SRM SIS were obtained.

Genome of the Single Human Chromosome 18 as a "Gold Standard" for Its Transcriptome.

The cutoff level applied in sequencing analysis varies according to the sequencing technology, sample type, and study purpose, which can largely affect the coverage and reliability of the data obtained. In this study, we aimed to determine the optimal combination of parameters for reliable RNA transcriptome data analysis. Toward this end, we compared the results obtained from different transcriptome analysis platforms (quantitative polymerase chain reaction, Illumina RNASeq, and Oxford Nanopore Technologies MinION) for the transcriptome encoded by human chromosome 18 (Chr 18) using the same sample types (HepG2 cells and liver tissue). A total of 275 protein-coding genes encoded by Chr 18 was taken as the gene set for evaluation. The combination of Illumina RNASeq and MinION nanopore technologies enabled the detection of at least one transcript for each protein-coding gene encoded by Chr 18. This combination also reduced the probability of false-positive detection of low-copy transcripts due to the simultaneous confirmation of the presence of a transcript by the two fundamentally different technologies: short reads essential for reliable detection (Illumina RNASeq) and long-read sequencing data (MinION). The combination of these technologies achieved complete coverage of all 275 protein-coding genes on Chr 18, identifying transcripts with non-zero expression levels. This approach can improve distinguishing the biological and technical reasons for the absence of mRNA detection for a given gene in transcriptomics.

Pressure tolerance of deep-sea enzymes can be evolved through increasing volume changes in protein transitions: a study with lactate dehydrogenases from abyssal and hadal fishes.

We explore the principles of pressure tolerance in enzymes of deep-sea fishes using lactate dehydrogenases (LDH) as a case study. We compared the effects of pressure on the activities of LDH from hadal snailfishes Notoliparis kermadecensis and Pseudoliparis swirei with those from a shallow-adapted Liparis florae and an abyssal grenadier Coryphaenoides armatus. We then quantified the LDH content in muscle homogenates using mass-spectrometric determination of the LDH-specific conserved peptide LNLVQR. Existing theory suggests that adaptation to high pressure requires a decrease in volume changes in enzymatic catalysis. Accordingly, evolved pressure tolerance must be accompanied with an important reduction in the volume change associated with pressure-promoted alteration of enzymatic activity ( ΔVPP∘ ). Our results suggest an important revision to this paradigm. Here, we describe an opposite effect of pressure adaptation-a substantial increase in the absolute value of ΔVPP∘ in deep-living species compared to shallow-water counterparts. With this change, the enzyme activities in abyssal and hadal species do not substantially decrease their activity with pressure increasing up to 1-2 kbar, well beyond full-ocean depth pressures. In contrast, the activity of the enzyme from the tidepool snailfish, L. florae, decreases nearly linearly from 1 to 2500 bar. The increased tolerance of LDH activity to pressure comes at the expense of decreased catalytic efficiency, which is compensated with increased enzyme contents in high-pressure-adapted species. The newly discovered strategy is presumably used when the enzyme mechanism involves the formation of potentially unstable excited transient states associated with substantial changes in enzyme-solvent interactions.