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.

MS Identification of Blood Plasma Proteins Concentrated on a Photocrosslinker-Modified Surface.

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.

Workability of mRNA Sequencing for Predicting Protein Abundance.

Transcriptomics methods (RNA-Seq, PCR) today are more routine and reproducible than proteomics methods, i.e., both mass spectrometry and immunochemical analysis.

Linking Clinical Blood Metabogram and Gut Microbiota.

Recently, a clinical blood metabogram was developed as a fast, low-cost and reproducible test that allows the implementation of metabolomics in clinical practice. The components of the metabogram are functionally related groups of blood metabolites associated with humoral regulation, the metabolism of lipids, carbohydrates and amines, lipid intake into the organism, and liver function, thereby providing clinically relevant information. It is known that the gut microbiota affects the blood metabolome, and the components of the blood metabolome may affect the composition of the gut microbiota.

Analysis of Single Biomacromolecules and Viruses: Is It a Myth or Reality?

The beginning of the twenty-first century witnessed novel breakthrough research directions in the life sciences, such as genomics, transcriptomics, translatomics, proteomics, metabolomics, and bioinformatics. A newly developed single-molecule approach addresses the physical and chemical properties and the functional activity of single (individual) biomacromolecules and viral particles. Within the alternative approach, the combination of "single-molecule approaches" is opposed to "omics approaches".

Blood Plasma Proteome: A Meta-Analysis of the Results of Protein Quantification in Human Blood by Targeted Mass Spectrometry.

A meta-analysis of the results of targeted quantitative screening of human blood plasma was performed to generate a reference standard kit that can be used for health analytics. The panel included 53 of the 296 proteins that form a “stable” part of the proteome of a healthy individual; these proteins were found in at least 70% of samples and were characterized by an interindividual coefficient of variation <40%. The concentration range of the selected proteins was 10−10−10−3 M and enrichment analysis revealed their association with rare familial diseases.

Gene-centric coverage of the human liver transcriptome: QPCR, Illumina, and Oxford Nanopore RNA-Seq.

It has been shown that the best coverage of the HepG2 cell line transcriptome encoded by genes of a single chromosome, chromosome 18, is achieved by a combination of two sequencing platforms, Illumina RNA-Seq and Oxford Nanopore Technologies (ONT), using cut-off levels of FPKM > 0 and TPM > 0, respectively. In this study, we investigated the extent to which the combination of these transcriptomic analysis methods makes it possible to achieve a high coverage of the transcriptome encoded by the genes of other human chromosomes. A comparative analysis of transcriptome coverage for various types of biological material was carried out, and the HepG2 cell line transcriptome was compared with the transcriptome of liver tissue cells.

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/).

Prediction of Monomeric and Dimeric Structures of CYP102A1 Using AlphaFold2 and AlphaFold Multimer and Assessment of Point Mutation Effect on the Efficiency of Intra- and Interprotein Electron Transfer.

The three-dimensional structure of monomers and homodimers of CYP102A1/WT (wild-type) proteins and their A83F and A83I mutant forms was predicted using the AlphaFold2 (AF2) and AlphaFold Multimer (AFMultimer) programs, which were compared with the rate constants of hydroxylation reactions of these enzyme forms to determine the efficiency of intra- and interprotein electron transport in the CYP102A1 hydroxylase system. The electron transfer rate constants (), which determine the rate of indole hydroxylation by the CYP102A1 system, were calculated based on the distances (R) between donor-acceptor prosthetic groups (PG) FAD→FMN→HEME of these proteins using factor β, which describes an exponential decay from R the speed of electron transport (ET) according to the tunnelling mechanism. It was shown that the structure of monomers in the homodimer, calculated using the AlpfaFold Multimer program, is in good agreement with the experimental structures of globular domains (HEME-, FMN-, and FAD-domains) in CYP102A1/WT obtained by X-ray structural analysis, and the structure of isolated monomers predicted in AF2 does not coincide with the structure of monomers in the homodimer, although a high level of similarity in individual domains remains.

Comparative Metabolomic Study of Species with Different Lifespans.

The increase in life expectancy, leading to a rise in the proportion of older people, is accompanied by a prevalence of age-related disorders among the world population, the fight against which today is one of the leading biomedical challenges. Exploring the biological insights concerning the lifespan is one of the ways to provide a background for designing an effective treatment for the increase in healthy years of life. Untargeted direct injection mass spectrometry-based metabolite profiling of 12 species of with significant variations in natural lifespans was conducted in this research.

Does Proteomic Mirror Reflect Clinical Characteristics of Obesity?

Obesity is a frightening chronic disease, which has tripled since 1975. It is not expected to slow down staying one of the leading cases of preventable death and resulting in an increased clinical and economic burden. Poor lifestyle choices and excessive intake of "cheap calories" are major contributors to obesity, triggering type 2 diabetes, cardiovascular diseases, and other comorbidities.

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.

Revelation of Proteomic Indicators for Colorectal Cancer in Initial Stages of Development.

Colorectal cancer (CRC) at a current clinical level is still hardly diagnosed, especially with regard to nascent tumors, which are typically asymptotic. Searching for reliable biomarkers of early diagnosis is an extremely essential task. Identification of specific post-translational modifications (PTM) may also significantly improve net benefits and tailor the process of CRC recognition.

The "Missing" Proteome: Undetected Proteins, Not-Translated Transcripts, and Untranscribed Genes.

The Chromosome-centric Human Proteome Project aims at characterizing the expression of proteins encoded in each chromosome at the tissue, cell, and subcellular levels. The proteomic profiling of a particular tissue or cell line commonly results in a substantial portion of proteins that are not observed (the "missing" proteome). The concurrent transcriptome profiling of the analyzed tissue/cells samples may help define the set of untranscribed genes in a given type of tissue or cell, thus narrowing the size of the "missing" proteome and allowing us to focus on defining the reasons behind undetected proteins, namely, whether they are technical (insufficient sensitivity of protein detection) or biological (correspond to not-translated transcripts).

Challenges of the Human Proteome Project: 10-Year Experience of the Russian Consortium.

This manuscript collects all the efforts of the Russian Consortium, bottlenecks revealed in the course of the C-HPP realization, and ways of their overcoming. One of the main bottlenecks in the C-HPP is the insufficient sensitivity of proteomic technologies, hampering the detection of low- and ultralow-copy number proteins forming the "dark part" of the human proteome. In the frame of MP-Challenge, to increase proteome coverage we suggest an experimental workflow based on a combination of shotgun technology and selected reaction monitoring with two-dimensional alkaline fractionation.

Metabolomic diagnostics and human digital image.

The existing clinical laboratory practice has limitations in terms of specificity and sensitivity of diagnosis, making the introduction of new methods in medicine more topical. Application of 'omics' technologies, especially metabolomics, allows overcoming these limitations. The composition of blood metabolites reflects the physical state of an organism at the molecular level.

200+ Protein Concentrations in Healthy Human Blood Plasma: Targeted Quantitative SRM SIS Screening of Chromosomes 18, 13, Y, and the Mitochondrial Chromosome Encoded Proteome.

This work continues the series of the quantitative measurements of the proteins encoded by different chromosomes in the blood plasma of a healthy person. Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards (SRM SIS) and a gene-centric approach, which is the basis for the implementation of the international Chromosome-centric Human Proteome Project (C-HPP), were applied for the quantitative measurement of proteins in human blood plasma. Analyses were carried out in the frame of C-HPP for each protein-coding gene of the four human chromosomes: 18, 13, Y, and mitochondrial.

Increased Sensitivity of Mass Spectrometry by Alkaline Two-Dimensional Liquid Chromatography: Deep Cover of the Human Proteome in Gene-Centric Mode.

Currently, great interest is paid to the identification of "missing" proteins that have not been detected in any biological material at the protein level (PE1). In this paper, using the Universal Proteomic Standard sets 1 and 2 (UPS1 and UPS2, respectively) as an example, we characterized mass spectrometric approaches from the point of view of sensitivity (Sn), specificity (Sp), and accuracy (Ac). The aim of the paper was to show the utility of a mass spectra approach for protein detection.

The Gene-Centric Content Management System and Its Application for Cognitive Proteomics.

The Human Proteome Project is moving into the next phase of creating and/or reconsidering the functional annotations of proteins using the chromosome-centric paradigm. This challenge cannot be solved exclusively using automated means, but rather requires human intelligence for interpreting the combined data. To foster the integration between human cognition and post-genome array a number of specific tools were recently developed, among them CAPER, GenomewidePDB, and The Proteome Browser (TPB).

Quantitative assessment of betamethasone dual-acting formulation in urine of patients with rheumatoid arthritis and ankylosing spondylitis after single-dose intramuscular administration and its application to long-term pharmacokinetic study.

Quantitative evaluation and assessment of pharmacokinetic parameters of Diprospan (suspension for injection 7mg/mL (2mg+5mg/mL) of betamethasone) were performed in urine samples taken from patients with rheumatoid arthritis or ankylosing spondylitis for 28days after systemic intramuscular administration in routine clinical practice in an open-comparative prospective cohort study. The maximum betamethasone concentration was reached at day 4 of the follow-up; in some cases, β-phase of elimination of the drug was appeared at day 14 or at day 21 of the follow-up. The deferred β-phase elimination was likely a consequence of the physiological characteristics of the patients or of the influence of non-steroidal agents.

Why Are the Correlations between mRNA and Protein Levels so Low among the 275 Predicted Protein-Coding Genes on Human Chromosome 18?

In this work targeted (selected reaction monitoring, SRM, PASSEL: PASS00697) and panoramic (shotgun LC-MS/MS, PRIDE: PXD00244) mass-spectrometric methods as well as transcriptomic analysis of the same samples using RNA-Seq and PCR methods (SRA experiment IDs: SRX341198, SRX267708, SRX395473, SRX390071) were applied for quantification of chromosome 18 encoded transcripts and proteins in human liver and HepG2 cells. The obtained data was used for the estimation of quantitative mRNA-protein ratios for the 275 genes of the selected chromosome in the selected tissues. The impact of methodological limitations of existing analytical proteomic methods on gene-specific mRNA-protein ratios and possible ways of overcoming these limitations for detection of missing proteins are also discussed.

Post-translational modifications of FDA-approved plasma biomarkers in glioblastoma samples.

Liquid chromatography-tandem mass spectrometry was used to analyze plasma proteins of volunteers (control) and patients with glioblastoma multiform (GBM). A database search was pre-set with a variable post-translational modification (PTM): phosphorylation, acetylation or ubiquitination. There were no significant differences between the control and the GBM groups regarding the number of protein identifications, sequence coverage or number of PTMs.

State of the Art of Chromosome 18-Centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells.

A gene-centric approach was applied for a large-scale study of expression products of a single chromosome. Transcriptome profiling of liver tissue and HepG2 cell line was independently performed using two RNA-Seq platforms (SOLiD and Illumina) and also by Droplet Digital PCR (ddPCR) and quantitative RT-PCR. Proteome profiling was performed using shotgun LC-MS/MS as well as selected reaction monitoring with stable isotope-labeled standards (SRM/SIS) for liver tissue and HepG2 cells.

Targeted Quantitative Screening of Chromosome 18 Encoded Proteome in Plasma Samples of Astronaut Candidates.

This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20-47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18.