Biology subjects

Martinez del Val, A.

Publications and source records attributed to Martinez del Val, A..

9 recordsLinked to original sources

bioRxiv2026

EpiFlow: multidimensional single-cell epigenetic profiling by spectral flow cytometry

The epigenetic landscape of individual cells determines their identity and function, yet current methods for profiling chromatin modifications at single-cell resolution remain low-throughput, costly, or limited in parametric depth. Here we present EpiFlow, a spectral flow cytometry-based platform that enables the simultaneous quantification of 16 epigenetic markers, including histone post-translational modifications, DNA methylation, and hydroxymethylation, at the single-cell level. We demonstrate that EpiFlow is robust across species from yeast to mammals and resolves biologically meaningful epigenetic transitions during the cell cycle, stem cell differentiation, germinal centre B cell maturation, diabetic liver remodelling, and seizure-induced chromatin reprogramming. High-dimensional integration of EpiFlow data enables cell-type classification based solely on epigenetic profiles in liver, brain, blood, and cancer. Furthermore, EpiFlow detects on-target and off-target/indirect effects of epigenetic drugs in a high-throughput-compatible format. Collectively, these results establish EpiFlow as a broadly applicable platform for single-cell epigenetic analysis in basic, pharmaceutical, and translational research.

cell biology↗

bioRxiv2025

The Human Cell Line Phosphoproteome Atlas: A Deep Empirical Resource Revealing Kinase Activity Landscapes

Protein phosphorylation orchestrates cellular signaling and controls diverse biological processes, with its dysregulation driving diseases, notably cancer. Comprehensive, high-throughput phosphoproteomics remains limited by detection sensitivity, data completeness, and computational bottlenecks, especially in low-input settings. We present the deepest empirical human phosphoproteome resource to date, regrouping over 200,000 class I phosphosites across 33 diverse human cell lines, and demonstrate that this spectral library dramatically improves single-shot phosphoproteomics with 30-fold faster data processing compared to library-free approaches and enhanced confidence in phosphosite localization even from minimal sample input. Integrating proteome and phosphoproteome data, we developed a combined kinase activity score, revealing cell line- and cancer-specific signaling vulnerabilities, many correlating with drug sensitivity. This resource accelerates deep, reproducible phosphoproteomics, enabling systematic functional mapping of cellular signaling networks, and empowers precision oncology by highlighting actionable kinase targets in diverse cell states.

biochemistry↗

bioRxiv2024

Phosphoproteomics highlights complex resource management upon inflammatory stimulation of fibroblasts

The stimulation of cells by inflammatory mediators gives rise to intricate signaling cascades, inducing specific biological functions. The role of kinase activation for the establishment of specific inflammatory functions has only scarcely been established. A time-course analysis of human fetal fibroblasts stimulated with Interleukin-1{beta} (IL-1{beta}) and/or Dexamethasone (Dex) was conducted using mass spectrometry-based proteomics and phosphoproteomics in conjunction with lysolipid and oxylipin profiling. The IL-1{beta} induced proteome alterations indicated metabolic, transcriptional, and translational activation, including inflammatory marker proteins such as NOS1, THBS1, and STING1. The induction of mitochondrial proteins and the formation of numerous lysolipids indicated an increase in beta-oxidation. In addition to the NF-{kappa}B and STAT signaling pathways, which are characteristic of inflammatory activation, the MAP and AKT kinase signaling pathways were found to be strongly induced. Six hours after treatment, the observed signaling events exhibited a notable decline, nearly returning to their initial states after 24 hours. It is noteworthy that nearly all of these signaling activities were also observed in cells treated with Dex alone. Additionally, the proteome exhibited transient alterations, which included proteins otherwise characteristic of an inflammatory response, such as MMP3 and NFKB2. Activation of the kinase PIKFYVE was apparently specific for dexamethasone but constituted a minority of the total phosphorylation events. Only after 24 hours was the induction of proteins characteristic of glucocorticoids, such as TSC22D3 and MAOA, observed. The analysis of the effects of dexamethasone on the background of established inflammatory signaling verified the known inhibitory functions and resulted in the expression of anti-inflammatory proteins and oxylipins, but hardly affected the signaling events involving MAP and AKT kinases. In conclusion, this data demonstrates that a majority of inflammation-associated signaling events in fibroblasts needs to be attributed to resource and stress management rather than the establishment of specific inflammatory effector functions.

biochemistry↗

bioRxiv2024

Deciphering molecular mechanisms of synergistic growth reduction in kinase inhibitor combinations

In cancer treatment, the persistent challenge of unresponsiveness of certain patients to drugs or the development of resistance post-treatment remains a significant concern. Drug combinations that synergistically reduce tumor growth emerge as a promising avenue to address this issue. Here, we aimed to characterize the mechanism of action of two synergistic drug combinations that target PI3K together with MEK1 or with TAK1 and used time course measurements of phosphoproteomics and transcriptomics in response to single inhibitors and their combinations. Our analysis untangled those responses driven by single drugs and responses that were unique to the combinations. We observed a high overlap between single-drug responses and their combinations, suggesting that single-drug mechanisms dominate the mechanism of action of the combinations of the kinase inhibitors. Despite a high overlap, both drug combinations exhibited a synergistic modulation of several cell fate regulators found at the convergence points of the targeted pathways, including the key regulator of intrinsic apoptosis BCL2L11. Interestingly, the responses in both combinations were largely limited to the targeted pathways, namely PI3K/AKT and MAPKs, with very limited change of any other additional cell fate decision pathways. In addition, we observed a strong downregulation of nucleotide metabolism and tRNA biosynthesis uniquely in the combinations, which could be attributed to the reduced activity of mTOR and ATF4. Our approach provides insights into the molecular mechanisms affected by the PI3Ki-TAK1i and PI3Ki-MEKi combinations and can serve as a flexible framework for dissecting drug combination responses based on multi-omics measurements.

cancer biology↗

bioRxiv2023

Systematic optimization of automated phosphopeptide enrichment for high-sensitivity phosphoproteomics

Improving coverage, robustness and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography tandem mass spectrometry (LC-MS/MS) runs from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-beads phosphoproteomics sample preparation with focus on low input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample and loading buffer volumes, allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 {micro}g of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=159 HEIGHT=200 SRC="FIGDIR/small/568418v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@f455d9org.highwire.dtl.DTLVardef@130537eorg.highwire.dtl.DTLVardef@1b99287org.highwire.dtl.DTLVardef@42e72e_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2023

Narrow-window DIA: Ultra-fast quantitative analysis of comprehensive proteomes with high sequencing depth

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here, we present an ultra-fast scanning data-independent acquisition (DIA) strategy consisting on 2-Th precursor isolation windows, dissolving the differences between data-dependent and independent methods. This is achieved by pairing a Quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer that provides >200 Hz MS/MS scanning speed, high resolving power and sensitivity, as well as low ppm-mass accuracy. Narrow-window DIA enables profiling of up to 100 full yeast proteomes per day, or [~]10,000 human proteins in half-an-hour. Moreover, multi-shot acquisition of fractionated samples allows comprehensive coverage of human proteomes in [~]3h, showing comparable depth to next-generation RNA sequencing and with 10x higher throughput compared to current state-of-the-art MS. High quantitative precision and accuracy is demonstrated with high peptide coverage in a 3-species proteome mixture, quantifying 14,000+ proteins in a single run in half-an-hour. TeaserAccurate and precise label-free quantification with comprehensive proteome coverage using narrow-window DIA

systems biology↗

bioRxiv2022

Hybrid-DIA: Intelligent Data Acquisition for Simultaneous Targeted and Discovery Phosphoproteomics in Single Spheroids

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging when analyzing tiny sample amounts. We present a novel hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem MS scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmarked hybrid-DIA against state-of-the-art targeted MS methods (i.e. SureQuant) using EGF-stimulated HeLa cells and found the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiled the global phosphoproteome. To demonstrate the robustness, sensitivity and potential of hybrid-DIA, we profiled chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluated the difference of cancer cells in 2D vs 3D culture. Altogether, we showed that hybrid-DIA is the way-to-go method in highly sensitive phospho-proteomics experiments.

systems biology↗

bioRxiv2021

Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolution

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry (MS)-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmarked the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in-vitro in HeLa cells and in-vivo in mouse tissues. Finally, we investigated the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io.

systems biology↗

bioRxiv2019

Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition (DIA) without the need for spectral libraries

Quantitative phosphoproteomics has in recent years revolutionized understanding of cell signaling, but it remains a challenge to scale the technology for high-throughput analyses. Here we present a rapid and reproducible phosphoproteomics approach to systematically analyze hundreds of samples by fast liquid chromatography tandem mass spectrometry using data independent acquisition (DIA). To overcome the inherent issue of positional phosphopeptide isomers in DIA-based phosphoproteomics, we developed and employed an accurate site localization scoring algorithm, which is incorporated into the Spectronaut software tool. Using a library of synthetic phosphopeptides spiked-in to a yeast phosphoproteome in different ratios we show that it is on par with the top site localization score for data-dependent acquisition (DDA) based phosphoproteomics. Single-shot DIA-based phosphoproteomics achieved an order of magnitude broader dynamic range, higher reproducibility of identification and improved sensitivity and accuracy of quantification compared to state-of-the-art DDA-based phosphoproteomics. Importantly, direct DIA without the need of spectral libraries performed almost on par with analyses using specific project-specific libraries. Moreover, we implemented and benchmarked an algorithm for globally determining phosphorylation site stoichiometry in DIA. Finally, we demonstrate the scalability of the DIA approach by systematically analyzing the effects of thirty different kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that a large proportion of EGF-dependent phospho-regulation is mediated by a specific set of protein kinases.

biochemistry↗