Biology subjects

Zubarev, R.

Publications and source records attributed to Zubarev, R..

16 recordsLinked to original sources

bioRxiv2026

Peptide-to-protein data aggregation using Fisher's method improves target identification in chemical proteomics

Protein-level statistical tests in proteomics aimed at obtaining p-value are conventionally made on protein abundances aggregated from peptide data. This integral approach overlooks peptide-level heterogeneity and ignores important information coded in individual peptide data, while protein p-value can also be obtained by Fishers method of combining peptide p-values using chi-square statistics. Here we test this latter approach across diverse chemical proteomics datasets based on assessments of protein expression, solubility and protease accessibility. Using the top four peptides ranked by their p-values consistently outperformed protein-level analysis and avoided biases introduced by inclusion of deviant peptides or imputation of missing peptide values. Fishers method provides a simple and robust strategy, improving identification of regulated/shifted proteins in diverse proteomics assays.

bioinformatics↗

bioRxiv2026

Low-nanogram Fourier Transform Isotopic Ratio Mass Spectrometry of Proteins

Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet, trophic interactions, and metabolic fluxes, but conventional isotope ratio mass spectrometry requires milligram-scale samples, limiting its applicability to small or rare biological specimens. Fourier Transform Isotopic Ratio Mass Spectrometry (FT IsoR MS) enables amino acid-resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale. Here, we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics-style sample preparation. Using human HeLa cells cultured in 13C-glucose-enriched and control media, we show that reliable relative {delta}13C measurements can be obtained from as few as 50 cells, corresponding to <10 ng of total protein, with a precision of approximately {+/-}9{per thousand}. The observed amino acid-specific labeling patterns are metabolically coherent and consistent with bulk measurements, while smaller cell numbers ([≤]10 cells) do not yield statistically robust results. These findings establish the practical sensitivity threshold of FT IsoR MS at the low-nanogram level and demonstrate its suitability for isotope-resolved analyses of small cell populations, micro-organoids, and other low-input biological samples, thereby extending stable isotope analysis toward single-cell-scale applications.

biochemistry↗

bioRxiv2025

Stochastic resonance is behind single cell proteomics success

Stochastic resonance is a phenomenon of noise-aided detection of below-threshold signal at the expense of the linearity of signal quantification. Here we demonstrate that peptide signal from single cell proteomes multiplexed by tandem mass tag (TMT) with a carrier proteome is below the nominal detection threshold in the Orbitrap mass analyzer and is registered in the mass spectra largely due to stochastic resonance amplification. This amplification is of the order of 0.5-2.5 single cell equivalents. Consequently, spiking by the corresponding amount of a constant "pedestal" proteome lifts the signal of the analyte single cell proteome above the detection threshold, improving detection and largely restoring linearity of quantification.

biochemistry↗

bioRxiv2025

High-ratiO partiaL proteolysiS with carriER proteome (HOLSER) Enables Global Structure Profiling and Site-resolved Elucidation of Ligand-Protein Interactions

Understanding how cellular proteins interact with their environment, including endogenous and exogeneous molecules, is critical for elucidating mechanisms of cellular regulation and drug action. Partial proteolysis-based techniques offer peptide-level resolution of ligand-induced conformational changes but are limited by modest proteome coverage and depth, as well as sensitivity to the experimental conditions. To overcome these limitations, we developed higH ratiO partiaL proteolysiS with carriER proteome (HOLSER), an efficient workflow that features extended digestion time for reduced peptide yield variability as well as tandem mass tag multiplexing that includes full digests for enhanced proteome depth and sequence coverage as well as higher precision of peptide abundance measurements. We demonstrate HOLSER capabilities of probing structural changes on the scale of specific binding sites for kinase target mapping, individual protein domains for structural mapping of the FKBP-mTOR complex in response to rapamycin as well as global proteome structure profiling.

biochemistry↗

bioRxiv2025

Acute high-dose irradiation of human primary ovarian cells reveals a shift in transcriptomics profile and impairment in cell-cell adhesion

STUDY QUESTIONHow do human cortical (cPOCs) and medullary (mPOCs) primary ovarian cells respond to acute X-ray exposure? SUMMARY ANSWERAcute high-dose X-ray exposure causes a shift in cPOCs and mPOCs transcriptomic profiles and impairs significantly their cell-cell adhesion ability. WHAT IS KNOWN ALREADYRadiotherapy is a leading cancer treatment, due to its effectiveness in targeting malignant cells. However, it can also affect healthy cells, potentially causing organ dysfunction, among which ovaries. When targeted radiotherapy is not feasible, fertility preservation is recommended to avoid premature ovarian insufficiency. In addition, the effects of irradiation on ovarian somatic cells remain poorly understood. STUDY DESIGN, SIZE, DURATIONOvarian tissue was obtained from patients undergoing gender-affirming surgery at Karolinska University Hospital Huddinge, Sweden. The ovarian tissue was separated into cortex and medulla, then individually dissociated into single-cell suspensions using mechanical and enzymatic methods. Monolayer cultures from cPOCs and mPOCs were exposed to a single dose of 10 Gy X-ray irradiation or left unexposed as paired controls. Following irradiation, the cells were cultured at various time-points for further molecular and morphological evaluation. PARTICIPANTS/MATERIALS, SETTING, METHODSOvarian tissue from 8 patients (age 23-36 years) was used. Dissociated cPOCs and mPOCs were cultured to 80% confluence and irradiated with 10 Gy (1.33 Gy/min), with non-irradiated controls. Cellular ATP and mitochondrial activity were assessed, followed by immunofluorescence staining for canonical irradiation-induced effects in cells: DNA damage, apoptosis and cell cycle progression. Bulk RNA-sequencing was performed on controls and irradiated samples. Libraries were prepared using the Illumina Stranded mRNA Prep Ligation protocol and sequenced on Illumina NovaSeq6000 platform. Genes were considered to be differentially expressed under the cut-off of false discovery rate (FDR) < 0.05. Subsequently, affected biological pathway was predicted using all expressed genes ranked by log2 fold change again hallmark gene sets. To further investigate the potential upstream regulators, transcription factor enrichment analysis were performed based on DEGs. To assess changes at protein level, we mapped the proteomic profile using liquid chromatography-tandem mass spectrometry. Peptides were considered to be differentially expressed (DEPs) under the cut-off of p-value < 0.01. Finally, we measured the ability of cPOCs and mPOCs to form 3D aggregates after seeding irradiated and non-irradiated cells on Biosilk scaffolds. MAIN RESULTS AND THE ROLE OF CHANCEFollowing irradiation, ATP levels and mitochondrial activity in cPOCs and mPOCs were comparable to controls, indicating minimal irradiation impact on cell viability and proliferation. Immunofluorescence analysis confirmed the modulation of canonical pathways, such as DNA damage, apoptosis and cell cycle in both cPOCs and mPOCs. Transcriptomic analysis showed that cPOCs and mPOCs at 1 h post-irradiation clustered together with the related 1 h control. However, a shift in transcriptomic profile was observed after 4 h and even more after 24 h post-irradiation in both cPOCs and mPOCs. Gene set enrichment analysis (GSEA) indicated upregulation of the p53 pathway at 4 h and 24 h post-irradiation, alongside downregulation of MYC targets, E2F targets, the G2/M checkpoint and mTORC1 pathway. Gene pattern analysis showed irradiation-dependent trends related to extracellular matrix (ECM) organisation, p53-mediated apoptotic mechanisms and chromosome segregation during the 24 h period following irradiation. Additionally, transcription factor enrichment analysis based on DEGs suggested p53 and MYC as potential upstream regulators. On a proteomic level, DEPs associated with ECM organisation and cytoskeleton formation were detected at 4 and 24 h post-irradiation. Finally, X-ray exposure hindered the cell-cell adhesion ability of both cPOCs and mPOCs, leading to impaired formation of Silk-Ovarioids. LARGE SCALE DATAThe RNA sequencing count matrix is deposited in Gene Expression Omnibus (GEO) with accession number GSE291604. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD061796. The code used for the analysis can be found at https://github.com/tialiv/X-Ovary. LIMITATIONS, REASONS FOR CAUTIONThe ovarian tissue was obtained from gender-affirming surgery patients who received androgen treatment before removal. Even though unlikely, this hormonal treatment might influence ovarian environment and alter cellular response to irradiation. Additionally, in this study the impact of X-ray exposure was assessed on a monolayer cell model, thus limiting the extrapolation power of our results to ovary in vivo. Lastly, the impact of irradiation was focused on the somatic cell populations that are essential for ovarian function. Further studies are needed to investigate the effects of X-ray exposure on ovarian follicles and their function. WIDER IMPLICATIONS OF THE FINDINGSUnderstanding the roles of MYC, p53 and cell adhesion factors in response to irradiation could guide the development of future ovarian protective strategies. These findings lay the foundation for further studies on ovarian tissue protection and fertility preservation in cancer patients. STUDY FUNDING/COMPETING INTEREST(S)This work was funded by the European Unions HORIZON 2020 research and innovation programme (MATER) under the Marie Skodowska-Curie Actions (grant agreement No: 813707), the Estonian Research Council (grants PRG1076 and PSG608), the Orion Research Foundation sr personal grant, the Research grant from the Center for Innovative Medicine (CIMED) and the Karolinska Institutet Consolidator Grant.

cell biology↗

bioRxiv2025

Above-Filter Digestion Proteomics reveals drug targets and localizes ligand binding site

While a number of efficient chemical proteomics methods are available for determining protein targets of pharmaceutical drugs, each approach exhibits its own "blind spot" and development of complementary techniques is needed. Here, we introduce the Above-Filter Digestion Proteomics (AFDIP) approach based on the monitoring of the rate of trypsin digestion. Digestion rate is decreased at the site of ligand binding to its target protein, while other sites may simultaneously experience an increase the digestion rate. Molecular dynamics (MD) simulations revealed that this increase may be due to allosteric structural changes related to backbone flexibility. We showcase the utility of AFDIP for deconvoluting the targets of versatile drugs and metabolites. Like other techniques, AFDIP allows for a two-dimensional analysis, with the second dimension based on drug concentration. AFDIP exhibits structural resolution, identifying drug target binding sites within [≤]10 [A], and for larger proteins often [≤]5 [A] of the crystallography-determined binding sites. Compared to earlier introduced limited proteolysis approaches, AFDIP provides easier sample preparation, deeper proteome analysis and broader sequence coverage. AFDIP is expected to find its place among the most efficient chemical proteomics methods currently available.

biochemistry↗

bioRxiv2024

ThermoTargetMiner as a proteome integral solubility alteration target database for prospective drugs against lung cancer

Knowledge of the targets of therapeutic compounds is vital for understanding their action mechanisms and side effects, but such valuable data is seldom available. The multiple complementary techniques needed for comprehensive target characterization must combine data reliability with sufficient analysis throughput. Here, we leveraged the Proteome Integral Solubility Alteration (PISA) assay to characterize the targets of 67 approved and experimental compounds against two main lung cancer subtypes, which is now provided through the website https://thermotargetminer.serve.scilifelab.se/app/thermotargetminer. Novel target candidates (pro-targets) were found for 77% of the tested molecules. Comparison of the protein solubility shifts in lysate vs. living cells highlighted the targets directly interacting with the compounds. We verified that the drug PEITC exerts cytotoxicity through the inhibition of a pro-target PAFAH1B. As PISA is now joining the arsenal of fast and reliable target characterization techniques, the presented database, ThermoTargetMiner, will become a useful resource in lung cancer research.

biochemistry↗

bioRxiv2024

Silk-Ovarioids: Establishment and characterization of human ovarian primary cells 3D-model system

In vitro models that mimic ovaries are crucial for elucidating the biological mechanisms underlying follicle activation and growth. Three-dimensional (3D) systems are particularly relevant because they can replicate the heterogeneity and cell-cell communication between different ovarian cell types. However, complex models using human ovarian primary cells have not yet been established. In this study, we developed and characterized long-term cultured 3D models of primary ovarian somatic cells isolated from adult tissues, using Biosilk as a scaffold. We successfully established both ovarian cortex- and medulla-derived 3D systems, termed Silk-Ovarioids. The presence of key ovarian somatic cell types - including granulosa, stromal, endothelial, and perivascular cells - was confirmed by transcriptomics, proteomics, and immunostaining. Notably, Silk-Ovarioids exhibited the formation of a pro-angiogenic hypoxic core, as evidenced by the development of vessel-like structures after six weeks of culture. The Silk-Ovarioids demonstrated low intra-batch variability and long-term culture stability, underscoring their potential as a robust step towards creating a bioengineered, patient-specific artificial ovary.

cell biology↗

bioRxiv2024

Do light eaters live shorter lives? The case of ultralight Caenorhabditis elegans

The idea that ingesting heavy stable isotopes can increase longevity emerged shortly after the discovery of deuterium in the early 1930s and has been extensively tested since then on animals. Here we present the first experimental evidence for the opposite. Growing C. elegans on bacteria E. coli that are in turn fed on a diet depleted of heavy isotopes of C, H, N and O produced ultralight worms that grow and mature faster but have a shorter lifespan. Based on the differences in expression and solubility of proteins, we established an aging pseudo-time scale. Notably, the newly born ultralight worms appear to be significantly "younger" than their normal counterparts, while at day 10 they are significantly "older". Pathway analysis revealed involvement of mitochondria; analysis of reactive oxygen species (ROS) confirmed significant ROS overproduction in ultralight worms that increases further with age. These findings provide a new modality of affecting the lifespan in this important animal model of human diseases and aging.

biochemistry↗

bioRxiv2022

Brain Endothelial Cells Exposure to Malaria Parasites Links Type I Interferon Signalling to Antigen Presentation, Immunoproteasome Activation, Endothelium Disruption, and Cellular Metabolism

Cerebral malaria (CM) lethality is attributable to brain edema induction but the cellular mechanisms involving brain microvascular endothelium in CM pathogenesis are unexplored. Activation of the STING-INFb-CXCL10 axis in brain endothelial cells (BECs) is a prominent component of the innate immune response in cerebral malaria (CM) development in mouse models. Using a T cell-reporter system, we show that Type I IFN signaling in BECs exposed to Plasmodium berghei-infected erythrocytes (PbA-IE), functionally enhances MHC Class-I antigen presentation through gamma-interferon independent immunoproteasome activation and impacted the proteome functionally related to vesicle trafficking, protein processing, and folding and antigen presentation. In vitro assays show that Type I IFN signaling and immunoproteasome activation are also involved in the dysfunction of the endothelial barrier through disturbing gene expression in the Wnt/{beta}-catenin signaling pathway. We demonstrate that IE exposure induces a substantial increase in BECs glucose uptake while glycolysis blockade abrogates INFb secretion impairing immunoproteasome activation, antigen presentation, and Wnt/{beta}-catenin signaling. Metabolome analysis show that energy demand and production are markedly increased in BECs exposed to IE as revealed by enriched content in glucose and amino acid catabolites. In accordance, glycolysis blockade in vivo delayed the clinical onset of CM in mice. Our results unveiled that Type I IFN signaling and subsequent immunoproteasome activation in BECs contribute to enhanced antigen presentation and the impairment of the endothelial barrier function. We also show that Type I IFN signaling and its downstream effects are licensed by dramatic increase in glucose uptake impacting on energy metabolism pathways. This work substantiates the hypothesis that Type I IFN-immunoproteasome induction response in BECs contributes to CM pathology and fatality (1) by increasing antigen presentation to cytotoxic CD8+ T cells and (2) by promoting endothelial barrier dysfunction, favoring brain vasogenic edema. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/518027v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@859960org.highwire.dtl.DTLVardef@1186de4org.highwire.dtl.DTLVardef@d6b625org.highwire.dtl.DTLVardef@50751d_HPS_FORMAT_FIGEXP M_FIG C_FIG AUTHOR SUMMARYCerebral malaria is a severe clinical form of malaria infection that leads to respiratory arrest and death due to brain swelling. Disruption of blood vessels in the brain barrier is a hallmark of cerebral malaria development. However, the underlying pathological mechanisms are still unsettled. We explored the hypothesis that immune response of brain blood vessels to malaria infection is an initiator of inflammatory events leading to cerebral malaria. Our experiments unveiled that proinflammatory Type I interferon action increases the presentation of parasite molecules by brain blood vessel cells to cytotoxic immune cells and promotes brain vessel disruption. We found that these effects are determined by activation of protein degradation systems upon exposure of brain blood vessel cells to malaria parasite-infected erythrocytes. Our findings unveil a critical role of Type I interferon in brain blood vessels cells signaling in initiating immunopathology mechanisms associated to cerebral malaria development and suggest that blocking Type I interferon induction in the brain provides a path to prevent this disease.

immunology↗

bioRxiv2022

System-wide profiling by proteome integral solubility alteration assay of drug residence times for target characterization

Most drugs used in the clinic and drug candidates target multiple proteins, and thus detailed characterization of their efficacy targets is required. While current methods rely on quantitative measurements at thermodynamic equilibrium, kinetic parameters such as the residence time of a drug on its target provide a better proxy for efficacy in vivo. Here, we present Residence Time Proteome Integral Solubility Alteration (ResT-PISA) assay which provides monitoring temporal protein solubility profiles after drug removal ("off-curve") in cell lysate or intact cells, quantifying the lifetime of drug-target interaction. A compressed version of the assay measures the integral under the off-curve enabling the multiplexing of binding affinity and residence time assessments into a single proteomic analysis. We introduce a combined scoring system for three parametric dimensions to improve prioritization of targets. By providing complementary information to other characteristics of drug-target interaction, ResT-PISA approach will be useful in drug development and precision medicine.

biochemistry↗

bioRxiv2021

Abnormal (hydroxy)prolines deuterium content redefines hydrogen chemical mass

Analysing the {delta}2H in individual amino acids of proteins extracted from vertebrates, we unexpectedly found in some samples, notably bone collagen from seals, more than twice as much deuterium in proline and hydroxyproline residues than in seawater. This corresponds to at least four times higher {delta}2H than in any previously reported biogenic sample. We ruled out diet as a plausible mechanism for such anomalous enrichment. This finding puts into question the old adage that you are what you eat. SUMMARYThe chemical mass of hydrogen is defined as an interval from the lowest to the highest content of deuterium 2H, hydrogens heavy stable isotope. Measurements of the deviations {delta}2H in the deuterium content from the standard (ocean water, {delta}2H = 0{per thousand}) are used to characterise biological samples, such as animal bone collagen. The results are often interpreted in terms of the trophic level and diet of the animal as well as prevailing climate during its lifetime. The majority of the published bone collagen {delta}2H data fall into a narrow {delta}2H range limited to {+/-}100{per thousand}. Using novel analysis method, we unexpectedly found greatly higher {delta}2H values, up to 1500{per thousand}, in seal bone collagen. Such anomalous deuterium enrichment is detected only in two amino acid residues, proline and its derivative hydroxyproline, while other residues show much smaller {delta}2H values. Anomalously high {delta}2H values, albeit of lower magnitudes, are also found for these residues in other biological sources. This finding substantially expands the upper bound of the hydrogen chemical mass for biogenic sources. Since neither diet nor environment explain these mysteriously high enrichment levels amounting to more than twice deuterium content in sea water, our understanding of stable isotopes in nature, as well as the old adage "you are what you eat", are put in question.

biochemistry↗

bioRxiv2021

Single Cell Chemical Proteomics (SCCP) Interrogates the Timing and Heterogeneity of Cancer Cell Commitment to Death

Chemical proteomics studies the effects of drugs upon cellular proteome. Due to the complexity and diversity of tumors, the response of cancer cells to drugs is also heterogeneous, and thus proteome analysis at single cell level is needed. Here we demonstrate that single cell proteomics techniques have become quantitative enough to tackle the drug effects on the target proteins, enabling single cell chemical proteomics (SCCP). Using SCCP, we studied here the time-resolved response of individual adenocarcinoma A549 cells to anticancer drugs methotrexate, camptothecin and tomudex, revealing the early emergence of cellular subpopulations committed and uncommitted to death. As a novel and useful approach to exploring the heterogeneous response to drugs of cancer cells, SCCP may prove to be a "killer application" for single cell proteomics.

cancer biology↗

bioRxiv2021

A spindle and thread-mechanism unblocks translation of N-terminally disordered proteins

Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT* domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT*. In summary, we demonstrate how inducing co-translational folding via a molecular "spindle and thread" mechanism can overcome poor translation efficiency of partially disordered proteins.

biochemistry↗

bioRxiv2018

System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA)

Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins might change their thermal stability. SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.

biochemistry↗

bioRxiv2018

ProTargetMiner: A proteome signature library of anticancer molecules for functional discovery

We present a publicly available, expandable proteome signature library of anticancer molecules in A549 adenocarcinoma cells. Based on 287 proteomes affected by 56 drugs, the main dataset contains 7,328 proteins and 1,307,859 refined protein-drug pairs. By employing the specificity concept in partial least square modeling, deconvolution of drug targets and mechanistic proteins is achieved for most compounds, including some kinase inhibitors. We built the first protein co-regulation database that takes into account both protein expression and degradation. A surprising number of strong anti-correlations is found, underscoring the importance of protein repression in cell regulation. Our analysis uncovered a group of proteins with extremely steady expression which are likely essential for core cellular functions. These findings bring about deeper understanding of cell mechanics. Extension of the dataset to novel compounds will facilitate drug design. The introduced specificity concept and modeling scheme are beneficial in other analysis types as well.\n\nStatement of SignificanceProTargetMiner is the first of its kind library of proteome responses of human cancer cells to anticancer molecules. This expandable resource facilitates the deconvolution of drug targets, action mechanisms, and cellular effects. It reveals death modalities, uncovers protein co-regulation and anti-correlation networks and defines the \"untouchable\" proteome essential for core cellular functionalities.

cell biology↗