Biology subjects

Nemes, P.

Publications and source records attributed to Nemes, P..

13 recordsLinked to original sources

bioRxiv2026

Analyte-Class-Dependent Electrophoretic Organization Enables Single-Run Proteome-Metabolome Analysis by CE-ESI-MS

Dual proteome-metabolome measurements from limited samples typically require sample splitting or sequential analyses using electrospray ionization mass spectrometry (ESI-MS). Here we show that capillary electrophoresis (CE) can avoid that tradeoff by organizing predominantly singly charged small molecules and multiply charged peptides into partially resolved, analyte-class-dependent regions of migration time-m/z space. Leveraging this intrinsic electrophoretic organization together with charge- and m/z-resolved precursor selection, we developed a single-run CE-ESI-MS workflow that combines single-vial sample processing with class-resolved tandem MS acquisition. In a HeLa digest spiked with 17 amino acids, the integrated analysis detected all amino acids while preserving proteomic depth relative to a dedicated proteomics run, yielding 1,221 versus 1,227 cumulative protein groups. Applied to identified single Xenopus laevis blastomeres, the method provided matched readouts of 86 metabolite features together with 1,097 and 1,083 protein groups from D1.1 and V1.1 cells, respectively. The paired measurements resolved cell-type-dependent molecular differences and mapped protein and metabolite changes into shared pathway context. These results establish analyte-class-dependent electrophoretic organization coupled to class-resolved MS acquisition as an analytical basis for single-run proteome-metabolome analysis by CE-ESI-MS in material-limited samples.

biochemistry↗

bioRxiv2026

Proteome-Driven Phenotyping of Identified Single Neurons in Intact Brain Tissue by Aspiration Patch Proteomics

Single-cell proteomics has advanced rapidly, but direct proteome measurements from identified neurons in intact brain tissue remain difficult because most workflows require cell isolation and recent patch-based studies have emphasized whole-soma retrieval. Here we show that aspiration-based patch proteomics enables deep proteome profiling of identified single neurons directly in acute mouse brain slices. We combined fluorescence-guided patch-clamp microsampling, minimal-loss bottom-up proteomics, and high-sensitivity capillary electrophoresis-timsTOF mass spectrometry to analyze partial somal aspirates from dopaminergic, parvalbumin, and serotonergic neurons in situ. The workflow identified more than 1,000 proteins from single-neuron samples under optimized conditions while consuming only about 0.25% of the processed digest per analysis. These proteomes were sufficient to separate biological replicates by neuronal phenotype, distinguish neuronal subtypes on the basis of protein expression alone, and define a conserved somal proteome shared across neuronal classes. Our results establish that controlled aspiration of partial somal material can support proteome-driven phenotyping without whole-soma retrieval, cell dissociation, or loss of native tissue context. Aspiration patch proteomics therefore provides an accessible route for subtype-level proteome phenotyping in intact brain tissue.

neuroscience↗

bioRxiv2026

Interface-Resolved Proteomics of Cell-Cell Membranes Reveals Early Spatial Polarity in a Vertebrate Embryo

Cell-cell membrane interfaces are central sites of adhesion, signaling, and polarity establishment, yet they have remained inaccessible to proteome-wide analysis as discrete analytical units. Here, we report an interface-resolved proteomics workflow that isolates intact intercellular membrane segments from single, identified blastomeres and quantitatively profiles their protein composition. Using a microdissection-enabled strategy combined with optimized mild-detergent extraction and high-sensitivity high-resolution mass spectrometry, we achieve deep coverage of low-input membrane samples, identifying [~]3,000 proteins per interface type, including over 100 annotated plasma-membrane proteins. Applying this approach to defined dorsal-dorsal, dorsal-ventral, and ventral-ventral cell-cell interfaces in a 16-cell chordate embryo model, Xenopus laevis, reveals reproducible, interface-specific proteomic signatures that distinguish neighboring membrane contacts along the primary body axis. Region-enriched proteins include regulators of membrane trafficking, signaling, cytoskeletal organization, and metabolic pathways linked to early dorsal-ventral patterning. These results demonstrate that intercellular membrane interfaces exhibit molecular polarity at early developmental stages and establish interface-resolved proteomics as a general strategy for mapping spatially organized biochemical activities at cell-cell contacts.

developmental biology↗

bioRxiv2025

Rapid CE-MS with Real-Time Eco-AI Resolves Proteomic Heterogeneity Among Single Human Neutrophils

Single-cell proteomics by mass spectrometry is advancing rapidly, yet throughput and sensitivity remain limiting--particularly for small, protein-poor cell types such as neutrophils. As the most abundant circulating leukocytes in humans, neutrophils are central to immune defense and inflammation, but their proteomes comprehensive single-cell level characterization has only concurrently been reported1 and remains limited. Here, we introduce a rapid capillary electrophoresis-mass spectrometry (CE-MS) platform, which integrates electrophoresis-correlative real-time data acquisition with sub-7-minute separations and artificial intelligence (AI)-based data processing software to achieve deep, high-throughput profiling. Using single-cell-equivalent HeLa digests, the Rapid Eco-AI platform identified [~]1,350 proteins from 300 pg and [~]835 proteins from 75 pg of input-- approaching the complexity of a mammalian cell proteome. Applied to freshly isolated human neutrophils, the workflow identified 151 proteins from [~]2 pg of material, [~]3% of the total cell proteome. Analysis of 13 individual cells revealed marked functional heterogeneity across pathways of degranulation, neutrophil extracellular trap (NET) formation, chemotaxis, and innate immunity, with hierarchical clustering resolving at least four distinct proteomic subtypes. These results establish Rapid Eco-AI as a sensitive, scalable, and broadly applicable CE-MS approach for immune-cell phenotyping at single-cell and subcellular resolution, facilitating new research opportunities in systems immunology and clinical proteomics.

developmental biology↗

bioRxiv2025

Subcellular Mass Spectrometry Reveals Proteome Remodeling in an Asymmetrically Dividing (Frog) Embryonic Stem Cell

Subcellular proteomics holds the potential to reveal the molecular architecture of cellular processes with unprecedented spatial resolution. Performing these analyses deeply, at the level of hundreds to thousands of proteins in subcellular resolution, is a high and still unmet technical need. Here, we advance microprobe capillary electrophoresis-mass spectrometry (CE-MS) to achieve deep proteomic coverage--quantifying over 1,000 proteins within opposing poles of an asymmetrically dividing embryonic stem cell (blastomere). We integrated CE-electrospray ionization (CE-ESI) with trapped ion mobility spectrometry time-of-flight (timsTOF) MS, implementing data-independent acquisition (DIA) via parallel accumulation-serial fragmentation (diaPASEF). This CE-diaPASEF workflow identified 1,035 proteins from [~]200 pg of proteome digest, equivalent to [~]80% of the HeLa cells content, with high reproducibility (coefficient of variation <15% across technical triplicates). With microprobe sampling, this technology quantified 808 to 1,022 proteins in opposing poles of a dorsal-animal (D1) blastomere in the 8-cell Xenopus laevis embryo. Comparative proteomic analysis of the D1 blastomere and its descendants--the dorsal-animal-midline (D11) and dorsal-animal-lateral (D12) cells--revealed diverse molecular outcomes of asymmetric division: some protein profiles remained conserved, while others underwent significant or even reversed changes as these lineages descended into neural tissue and epidermal trajectories. Ultraviolet light-induced ventralization was performed to help disentangle subcellular gradients from dorsal-ventral patterning. Collectively, this work establishes microprobe CE-diaPASEF as a powerful platform for deep subcellular proteomics, enabling new insights into spatial proteome organization during key developmental processes.

developmental biology↗

bioRxiv2025

Live Eco-AI, Electrophoresis-Correlative Data-Dependent Acquisition with Artificial Intelligence-Based Data Processing Democratizes Single-Cell Mass Spectrometry Proteomics

Single-cell mass spectrometry (MS) recently emerged with unprecedented sensitivity to characterize cellular proteomes. Still, limited access to sensitive and fast mass spectrometers impedes its broad adoption, due chiefly to budget constraints. Here, we democratize single-cell MS to a budget-conscious alternative with contemporary performance. A custom-built inexpensive capillary electrophoresis (CE) platform electrophoresis-correlatively (Eco) sorted peptide m/z signals. Detection re-employed a decade-old, slower yet affordable quadrupole-quadrupole-orbitrap (Q-Exactive Plus, QE+, Thermo). Data-dependent acquisition (DDA) with narrow, 1.6-to-4 Th isolation tracked the m/z trends in [~]100% efficiency, outperforming the data-independent reference. The ensuing chimeric spectra were deciphered using artificial intelligence (AI) via CHIMERYS (Thermo). Live Eco-AI augmented our QE+ to [~]15 peptides/spectrum, on par with more advanced analyzers (Lumos and Exploris compared). 1 ng HeLa proteome digest gave 2,142 proteins, topping 969 on a contemporary LC Qq-OT-ion trap reference (Fusion Lumos, Thermo). From [~]250 pg, approximately a cell, 1,799 HeLa-proteins were detected in <15-min-effective electrophoresis. To establish proof of principle, Live Eco-AI was employed to profile 1,524 proteins among differentially fated single stem cells (50-to-75 micrometer diameter) in the Xenopus laevis (frog) blastula. The quantitative data revealed previously unknown proteome reorganization during differentiation into the dorsal and ventral lineages.

cell biology↗

bioRxiv2024

Electrophoresis-Correlative Ion Mobility Deepens Single-cell Proteomics in Capillary Electrophoresis Mass Spectrometry

Detection of trace-sensitive signals is a current challenge is single-cell mass spectrometry (MS) proteomics. Separation prior to detection improves the fidelity and depth of proteome identification and quantification. We recently recognized capillary electrophoresis (CE) electrospray ionization (ESI) for ordering peptides into mass-to-charge (m/z)-dependent series, introducing electrophoresis-correlative (Eco) data-independent acquisition. Here, we demonstrate that these correlations based on electrophoretic mobility ({micro}ef) in the liquid phase are transferred into the gas phase, essentially temporally ordering the peptide ions into charge-dependent ion mobility (IM, 1/K0) trends ({rho} > 0.97). Rather than sampling the entire IM region broadly, we pursued these predictable correlations to schedule narrower frames. Compared to classical ddaPASEF, Eco-framing significantly enhanced the resolution of IM on a trapped ion mobility mass spectrometer (timsTOF PRO). This approach returned [~]50% more proteins from HeLa proteome digests approximating to one-to-two cells, identifying [~]962 proteins from [~]200 pg in <20 min of effective electrophoresis, without match-between-runs. As a proof of principle, we deployed Eco-ddaPASEF on 1,157 proteins by analyzing <4% of the total proteome in single, yolk-laden embryonic stem cells ([~]80-{micro}m) that were isolated from the animal cap of the South African clawed frog (Xenopus laevis). Quantitative profiling of 9 different blastomeres revealed detectable differences among these cells, which are normally fated to form the ectoderm but retain pluripotentiality. Eco-framing effectively deepens the proteome sensitivity in IMS using ddaPASEF, raising the possibility of a proteome-driven classification of embryonic cell differentiation.

developmental biology↗

bioRxiv2024

The 15-min (Sub)Cellular Proteome

Single-cell mass spectrometry (MS) opens a proteomic window onto the inner workings of cells. Here, we report the discovery characterization of the subcellular proteome of single, identified embryonic cells in record speed and molecular coverage. We integrated subcellular capillary microsampling, fast capillary electrophoresis (CE), high-efficiency nano-flow electrospray ionization, and orbitrap tandem MS. In proof-of-principle tests, we found shorter separation times to hinder proteome detection using DDA, but not DIA. Within a 15-min effective separation window, CE data-independent acquisition (DIA) was able to identify 1,161 proteins from single HeLa-cell-equivalent ([~]200 pg) proteome digests vs. 401 proteins by the reference data-dependent acquisition (DDA) on the same platform. The approach measured 1,242 proteins from subcellular niches in an identified cell in the live Xenopus laevis (frog) embryo, including many canonical components of organelles. CE-MS with DIA enables fast, sensitive, and deep profiling of the (sub)cellular proteome, expanding the bioanalytical toolbox of cell biology. Authorship ContributionsP.N. and B.S. designed the study. L.R.P. collected the X. laevis cell aspirates. B.S. prepared and measured the samples. B.S. and P.N. analyzed the data and interpreted the results. P.N. and B.S. wrote the manuscript. All the authors commented on the manuscript.

cell biology↗

bioRxiv2023

Mapping protein-exopolysaccharide binding interaction in Staphylococcus epidermidis biofilms by live cell proximity labeling

Bacterial biofilms consist of cells encased in an extracellular polymeric substance (EPS) composed of exopolysaccharides, extracellular DNA, and proteins that are critical for cell-cell adhesion and protect the cells from environmental stress, antibiotic treatments, and the host immune response. Degrading EPS components or blocking their production have emerged as promising strategies for prevention or dispersal of bacterial biofilms, but we still have little information about the specific biomolecular interactions that occur between cells and EPS components and how those interactions contribute to biofilm production. Staphylococcus epidermidis is a leading cause of nosocomial infections as a result of producing biofilms that use the exopolysaccharide poly- (1[->]6)-{beta}-N-acetylglucosamine (PNAG) as a major structural component. In this study, we have developed a live cell proximity labeling approach combined with quantitative mass spectrometry-based proteomics to map the PNAG interactome of live S. epidermidis biofilms. Through these measurements we discovered elastin-binding protein (EbpS) as a major PNAG-interacting protein. Using live cell binding measurements, we found that the lysin motif (LysM) domain of EbpS specifically binds to PNAG present in S. epidermidis biofilms. Our work provides a novel method for the rapid identification of exopolysaccharide-binding proteins in live biofilms that will help to extend our understanding of the biomolecular interactions that are required for bacterial biofilm formation.

microbiology↗

bioRxiv2023

Cell Lineage-Guided Microanalytical Mass Spectrometry Reveals Increased Energy Metabolism and Reactive Oxygen Species in the Vertebrate Organizer

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been limited to transcripts and some proteins due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been discovered to induce the patterning of the central nervous system. Molecular screens on the tissue have identified several genes, such as goosecoid, chordin, and noggin, with independent ability to establish a body axis. A comprehensive study of proteins and metabolites produced in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the rest of the embryo using liquid chromatography high-resolution mass spectrometry (HRMS). Quantification of [~]4,600 proteins and a panel of metabolites documented differential expression for [~]450 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation (OXPHOS) and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the Organizer. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO tissue. Chemical perturbation of the redox gradient affected mesoderm involution during early tissue movements of gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.

developmental biology↗

bioRxiv2021

Patch-Clamp Proteomics of Single Neuronal Somas in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry

Understanding of the relationship between cellular function and molecular composition holds a key to next-generational therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semi-routine measurement of single-cell genomes and transcriptomes, but analytical tools are scarce for detecting diverse proteins in tissue-embedded cells. To bridge this gap for neuroscience research, we report the integration of patch-clamp electrophysiology with subcellular shot-gun proteomics by high-resolution mass spectrometry (HRMS). Recording of electrical activity permitted identification of dopaminergic neurons in the substantia nigra pars compacta. Ca. 20-50% of the neuronal soma content, containing an estimated 100 pg of total protein, was aspirated into the patch pipette filled with ammonium bicarbonate. About ~1 pg of somal protein, or ~0.25% of the total cellular proteome, was analyzed on a custom-built capillary electrophoresis (CE) high-resolution mass spectrometer (HRMS). A series of experiments were conducted to systematically enhance detection sensitivity through refinements in sample processing and detection, allowing us to quantify ~275 different proteins from somal aspirate-equivalent protein digests. From single neurons, patch-clamp proteomics of the soma quantified 91, 80, and 95 different proteins from 3 different dopaminergic neurons, or 157 proteins in total. Quantification revealed detectable proteomic differences between the somal protein samples. Analysis of canonical knowledge predicted rich interaction networks between the proteins. The integration of patch-clamp electrophysiology with subcellular CE-HRMS proteomics expands the analytical toolbox of neuroscience.

neuroscience↗

bioRxiv2021

A Data-Dependent Acquisition Ladder for Ultrasensitive (Neuro)Proteomics

Measurement of broad types of proteins from a small number of cells to single cells would help to better understand the nervous system but requires significant leaps in high-resolution mass spectrometry (HRMS) sensitivity. Microanalytical capillary electrophoresis electrospray ionization (CE-ESI) offers a path to ultrasensitive proteomics by integrating scalability with sensitivity. We report here a data acquisition strategy that expands the detectable and quantifiable proteome in trace amounts of digests using CE-ESI-HRMS. Data-dependent acquisition (DDA) was programmed to progressively exclude high-intensity peptide signals during repeated measurements. These nested experiments formed rungs of our "DDA ladder." The method was tested for replicates analyzing ~500 pg of protein digest from cultured hippocampal (primary) neurons (mouse), which estimates to the total amount of protein from a single neuron. Analysis of net amounts approximating to ~10 neurons identified 428 nonredundant proteins (415 quantified), an ~35% increase over traditional DDA. The identified proteins were enriched in neuronal marker genes and molecular pathways of neurobiological importance. The DDA ladder deepens the detectable proteome from trace amounts of proteins, expanding the analytical toolbox of neuroscience.

neuroscience↗

bioRxiv2021

In Vivo Subcellular Mass Spectrometry Enables Proteo-Metabolomic Single-cell Systems Biology in a Chordate Embryo Developing to a Normally Behaving Tadpole (X. laevis)

We present the first example of in vivo high-resolution mass spectrometry (HRMS) for subcellular molecular systems biology of proteins and metabolites. With light microscopy, we identified the left-dorsal and left-ventral animal cells in cleavage-stage non-sentient Xenopus laevis embryos. Using precision-translated fabricated microcapillaries, the subcellular content of each cell was double-probed, each time collecting <5% of cell volume (~10 nL) swiftly (<5 s/event). The proteins and metabolites were analyzed by custom-built ultrasensitive capillary electrophoresis electrospray ionization employing Orbitrap and time-of-flight HRMS. Label-free detection of ~150 metabolites (57 identified) and 738 proteins found proteo-metabolomic networks with differential quantitative activities between the cell types. Spatially and temporally scalable sampling the technology preserved the integrity of the analyzed cells, the neighboring cells, and the embryo. 95% of the analyzed embryos developed into sentient tadpoles that were indistinguishable from their wild-type siblings based on anatomy and visual function in a background color preference assay.

developmental biology↗