Biology subjects

Pirog, A.

Publications and source records attributed to Pirog, A..

6 recordsLinked to original sources

bioRxiv2026

Novel native serum peptidomics workflow enables the discovery of circulating subtype-specific peptide biomarkers in acute ischemic and haemorrhagic stroke

Novel serum peptidomics offers a direct insight into proteolytic activity, tissue injury, and systemic signaling. Nevertheless, existing workflows suffer from low peptide yields, low throughput, and limited recovery of low-abundance species. Here we present a native serum peptidomics protocol that integrates mild acid treatment, solid-phase extraction with molecular weight cutoff filtration and data-independent acquisition mass spectrometry (DIA-MS). The protocol requires less than 100 {micro}l of serum or plasma, is completed within hours, time-cost-effective and compatible with 96-well formats without specialized equipment. Applied to a proof-of-concept cohort of patients with acute ischemic stroke (AIS), intracranial haemorrhage (ICH), and healthy controls, the workflow identified over 12,000 peptides, exceeding the three-fold threshold of existing peptidomics approaches. DIA-MS analysis across independent batches demonstrated 78-83% peptide overlap and consistent fold-change directionality. We further introduce peptide locus analysis, which aggregates overlapping peptides within defined protein regions. This approach revealed bidirectional regulation within individual precursor proteins such as the fibrinogen alpha chain (FIBA), resolving intraprotein proteolytic dynamics. Three candidate peptides from TYB4, CO4B, and ITIH4 proteins accurately distinguished stroke subtypes and controls, while characteristic shifts in peptide physicochemical properties were observed across strokes. This workflow substantially advances the sensitivity, throughput, and biological resolution of serum peptidomics for quantitative multi-biomarker discovery, validation and its output promises effective implementation of AI/ML models aiming for new dimensions in diagnostics, prognostics, prediction and monitoring.

neuroscience↗

bioRxiv2026

Benchmarking five extracellular vesicle proteomics workflows, including low-input Exo-insert and Exo-SP3, for deep mass spectrometry profiling

Small extracellular vesicles (sEVs) are key mediators of intercellular communication, influencing diverse pathological processes, including cancer. While mass spectrometry (MS) has enabled the proteomic analysis of sEVs, sample preparation losses remain a critical bottleneck, particularly for scarce tissue-derived sEVs (Ti-EVs). Here, we systematically benchmark five proteomic workflows introducing Exo-insert, a novel single-vessel method, and Exo-SP3, across both Ti-Evs and cell culture-derived sEVs (CCM-EVs) at low input (0.5-4 {micro}g). Exo-insert and Exo-SP3 enable the identification of [~]1100 protein groups from as little as 0.5 {micro}g sEV input. Notably, optimal sample preparation for MS is source-dependent: Exo-insert and Exo-SP3 display divergent performance across sEV sources. Comparative DDA/DIA analyses establish sample preparation as the primary determinant of proteome recovery, offering a practical framework that matches workflows to sEV amounts and source-specific content for biomarker discovery.

cancer biology↗

bioRxiv2026

Mature tumoroids recapitulate clinically relevant drug response through extended 3D culture in PDAC

BackgroundDrug responses in pancreatic ductal adenocarcinoma (PDAC) vary sharply across in vitro culture formats, but most 2D-3D comparisons conflate microenvironmental cues with time-dependent cellular adaptation. As a result, conventional assays frequently overestimate drug efficacy and poorly reflect clinical pharmacology. Main findingsWe profiled MiaPaCa-2, PANC-1, and CFPAC-1 grown in an extracellular-matrix (ECM) hydrogel for 1-12 days, defining extended 3D cultures ([≥]10 days) as mature tumoroids, and quantified 72 h drug responses to a multi-class oncology panel using growth-rate (GR) metrics to normalize for proliferation across formats and durations. Prolonged 3D pre-culture induced broad tolerance, with typical 10-100x reductions in sensitivity to standards of care (5-fluorouracil, SN38, oxaliplatin, gemcitabine, paclitaxel), following a reproducible susceptibility hierarchy (MiaPaCa-2 > PANC-1 > CFPAC-1) after GR correction. In mature tumoroids, GR values closely approximated clinically observed plasma exposures (e.g., within <4x for 5-FU and <0.5x for gemcitabine), whereas 2D and short-term organoid assays markedly underestimated resistance, often by >100x, thereby overstating drug activity. Notably, CFPAC-1 exhibited increased sensitivity to SN38 and trametinib under mature-organoid conditions, demonstrating that microenvironmental conditioning can invert responses for selected mechanisms. Transcriptomic profiling revealed coordinated up-regulation of multiple ABC transporters with extended 3D residence, tracking resistance phenotypes across lines and implicating transporter-linked tolerance programs. SignificanceTogether, these data identify time-in-3D and the emergence of mature tumoroids as dominant, previously under-controlled determinants of PDAC pharmacology that both induce tolerance and unmask context-dependent vulnerabilities. We propose incorporating both short-term and mature-tumoroid screening arms into preclinical workflows, reporting pre-culture duration alongside GR-normalized effect sizes, and leveraging transporter-informed biomarkers to guide regimen prioritization and sequencing. This framework enhances physiological relevance, reproducibility, and translational fidelity in PDAC drug discovery.

cancer biology↗

bioRxiv2026

Pancreatic α-cells are required for nutrient homeostasis by regulating dynamic β-cell networks in islets

Pancreatic islets contain -, {beta}-, {gamma}- and {delta}-cells as sensors and actuators regulating glucose homeostasis. Despite the known importance of -cells, they are seemingly required for glucose tolerance only under metabolic stress. In an inducible model of -cell ablation in mice (GluDTR), glucose tolerance was considerably decreased by physiological addition of amino-acids mimicking meals. Analysis of islet {beta}-cell secretion and electrical activities using microelectrode arrays (MEA) detected only minor differences in GluDTR mice for glucose but revealed a major reduction upon addition of amino acids. Analysis of functional islet {beta}-cell networks by high density MEA revealed leading regions in different locations, a high degree of synchrony and the activation of large cell clusters. The characteristics of leading regions were preserved in GluDTR islets, but synchrony, cluster size and signal propagation speed were largely reduced. Thus, even without metabolic stress, -cells are required for nutrient homeostasis by regulating the dynamics of {beta}-cell networks. TeaserIslet -cells are required for meal tolerance by adjusting synchrony, cluster size and signal propagation of {beta}-cell networks.

physiology↗

bioRxiv2024

Universal toolset for mass spectrometric analysis of intracellular peptidome and small protein fraction

The analysis of native intracellular peptidome has gained significant attention in recent years. However, there is still a need for more knowledge regarding various sample preparation methods that facilitate efficient and reproducible recovery of peptides, which can then be analyzed using quantitative liquid chromatography-mass spectrometry. A similar situation exists in small proteome research, typically defined as polypeptides with masses of less than 100 amino acids, often too long for easy identification without enzymatic digestion. In this context, we describe a set of methods that involve simple denaturation and solid-phase extraction of polypeptides, applicable for isolating short intracellular polypeptides within the desired length range. Our work demonstrates the efficiency and reproducibility of these methods for quantitative analysis of the peptidome in mammalian cells. Additionally, we investigated the flexibility of adjusting the mass range through ultrafiltration. We have shown that these methods can be adapted for highly efficient enrichment and fractionation of small proteins, resulting in polypeptide isolates suitable for tryptic digestion and intact protein analysis. Moreover, we describe the use of freely available computational tools that can effectively manage the analysis of the resulting data. The research presented here will benefit the global scientific community in both fundamental (protein turnover, proteolytic processing, non-canonical open reading frames, etc.) and applied sciences (bioactive/neuro peptide discovery, precision medicine, vaccines, etc.), and other areas that could benefit from selective analysis of short native polypeptides.

cancer biology↗

bioRxiv2024

Continuous monitoring of glucose levels in vivo with a micro-organ based microfluidic biosensor

Continuous monitoring of glucose levels has improved diabetes therapy. Current approaches rely on enzyme-linked electrochemical probes but do not allow a fully autonomous artificial pancreas. In contrast, monitoring the activity of a few electrogenic pancreatic islets in a biosensor may harness the computational power of the different endocrine cell types in the micro-organ, shaped for nutrient detection during evolution, and provide a more appropriate read-out. Extracellular electrophysiology captures slow potentials (SPs), which reflect coupled islet {beta}-cell activity and is thus a method of choice for long-term monitoring of native islet activity in vitro. We have now developed a microfluidic microelectrode chip containing a few islets and linked to interstitial fluids in live rats by subcutaneous microdialysis. The electrical activity in terms of slow potentials monitored by this biosensor reacts ex vivo proportionally to glucose levels off-line in serum or dialysed interstitial fluid. On-line monitoring in vivo reveals an excellent correlation between islet slow potential frequency, and to a lesser degree to slow potential amplitudes, to glucose concentrations with little variation between animals. The microorgan-based biosensor harness multiple parameters in vivo and provides a read-out closer to physiology. This demonstrates the usefulness of such biosensors for sensor-based therapy of diabetes.

physiology↗