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Multiplexed single cell proteomics of hypoxia in HEK293 expressing PIP-FUCCI

ABSTRACT: This study aimed to characterize temporal proteome dynamics of hypoxia using single-cell mass spectrometry-based proteomics. HEK293 cells expressing PIP-FUCCI cell cycle markers were grown in suspension culture. Cells were exposed to hypoxia and temporally sampled for single-cell sorting into 384-well plates. Samples were processed and multiplexed using TMT labeling according to the SCoPE2 workflow and analyzed using the RETICLE acquisition method. In parallel, single-cell RNA-seq data were also collected. These datasets were used together to disentangle the effects of hypoxia from cell cycle progression and to construct joint transcriptional and translational trajectories along the hypoxia response.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Craig Barry

LAB HEAD: Craig Barry

PROVIDER: PXD053053 | Pride | 2026-02-16

REPOSITORIES: Pride

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	20220924_T4_001.raw	Raw
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	20220924_T4_005.raw	Raw

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Publications

Resolving Single-Cell Gene Expression by Pseudotemporal Integration of Transcriptomic and Proteomic Datasets.

Barry Craig P CP Talbo Gert H GH Beauglehole Aiden A Ovchinnikov Dmitry D Munro Trent T Mahler Stephen S Baker Kym K Nielsen Lars K LK Mercer Tim R TR Marcellin Esteban E

Molecular & cellular proteomics : MCP 20251127 1

Single-cell omics technologies, such as single-cell RNA-Seq and single-cell proteomics, offer unprecedented insights into cellular heterogeneity and dynamic regulatory processes. However, integrating these data types to construct comprehensive transcription-translation profiles remains challenging because of their distinct and complex behaviors. This study presents a novel approach using pseudotemporal cell ordering to integrate single-cell RNA-Seq and single-cell proteomics by mass spectrometry ...[more]

PMID: 41317903

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Quantitative Single-Cell Proteomics as a Tool to Characterize Cellular Hierarchies

Project description:Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying approximately 1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we developed a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a solid foundation for implementing global single-cell proteomics studies across the world.

2021-05-06 | PXD020586 | Pride

Hypoxia induces histone clipping and H3K4me3 loss in neutrophil progenitors resulting in long-term impairment of neutrophil immunity

Project description:A deeper understanding of the processes that sustain long-term reprogramming of rapidly turned-over cells in humans can offer unprecedented therapeutic potential, especially for immune cells like neutrophils, given their short lifespan in circulation and importance in both acute and chronic diseases. Hypoxia is a hallmark of a plethora of disease states and significantly shapes neutrophil effector functions. However, its potential contribution to long-term neutrophil reprogramming remains largely unexplored. We have observed persistent perturbations in newly formed circulating neutrophils’ phenotype and function in patients 3-6 months following hospitalisation with acute respiratory distress syndrome (ARDS), where poor oxygenation takes place. These lasting changes are associated with widespread loss of H3K4me3 in genomic regions of relevance to neutrophil activities. Notably, the replication of these findings in healthy volunteers months following altitude induced hypoxaemia attributes a role to the oxygen deprivation as a driver for long-term neutrophil reprogramming. Mechanistically, mouse models of systemic hypoxia reveal deleterious outcomes for infection and show that persistent loss of H3K4me3 originates in proNeu and preNeu populations within the bone marrow and is linked to hypoxia-driven n terminal histone 3 clipping. Thus, we present novel evidence that acute adaptations to systemic hypoxia trigger central changes in newly formed neutrophil populations, resulting in long-term impairment of effector functions and placing hypoxia as a crucial orchestrator of sustained neutrophil reprogramming.

2025-09-22 | PXD065562 | Pride

Real-Time Search Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics

Project description:In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and we present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. Here we show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.

2022-03-14 | PXD029320 | Pride

Multi-modal single-cell sequencing of differentiating mESCs using Smart-seq3 and scATAC-seq

Project description:We report dynamics of X-chromosome upregulation (XCU) along X-chromosome inactivation (XCI) in mESCs as they differentiate into EpiSCs. F1 hybrid C57BL6/J × CAST/EiJ male and female mESCs were grown in serum/LIF conditions were differentiated using Fgf2 and Activin A for 1, 2, 4 and 7 days to induce random XCI in female cells. Multi-modal single-cell sequencing was performed using scATAC on nuclei and Smart-seq3 to assay chromatin accessibility and poly-A+ RNA expression, respectively. Allelic resolution is achieved using strain-specific SNPs in the data. We reveal dynamic balancing of X alleles as cells undergo XCI to compensate dosage imbalances between sexes as well as between X and autosomes. Furthermore, we reveal that female naïve mESCs with two active X chromosomes lack XCU on both alleles which has major implications for reprogramming studies. Finally, we estimate allelic transcriptional burst kinetics from the data and find that progressively increased burst frequencies underlies the XCU process.

2022-02-14 | E-MTAB-10714 | biostudies-arrayexpress

Single-cell proteomic landscape of the developing human brain

Project description:The label free single cell proteome profiling in developing human brain.

2025-12-05 | PXD071075 | Pride

ChIP-chip to investigate transcriptional control of hypoxic hyphal growth in the fungal pathogen Candida albicans

Project description:So far, there is no known regulatory circuits that mediate filamentation of the pathogenic yeast Candida albicans exclusively in response to hypoxia. In this study, we performed a quantitative analysis of gene deletion mutants from different collections of protein kinases and transcriptional regulators to identify specific regulator of the hypoxic filamentation. Our work uncovered two transcription factors, Ahr1 and Tye7, that act as prominent regulators of C. albicans filamentation specifically under hypoxia. In summary, we used genome-wide transcriptional profiling and promoter occupancy to characterize both Ahr1 and Tye7 regulons associated with the hypoxic filamentation in C. albicans.

2021-08-18 | E-MTAB-10883 | biostudies-arrayexpress

Crosslinking of Ly6a metabolically reprogram CD8 T cells for cancer immunotherapy.

Project description:To evaluate the downstream effects of anti-Ly6a crosslinking, we examined the effect of anti-Ly6a antibodies on CD8+ T cell activity in the presence and absence of target tumor cells by high-resolution mass spectrometry proteomic analyses. Spleen cells were isolated from gp10025–33 TCR transgenic mice and incubated for 3 days with IL-2, IFNα, and gp10025–33 peptide. Next, CD8+ T-cells were isolated and incubated overnight with anti-Ly6a antibody (or IgG with or without B16F10 melanoma cells. CD8+ T-cells were then sorted and subjected to proteomic analysis using high-resolution mass spectrometry

2024-08-04 | PXD050734 | Pride

scRNA-seq for mEpiSCs (epiblast stem cells) differentiated to posterior primitive streak (PPS) in vitro, in a monolayer

Project description:EpiSCs established from murine epiblast were differentiated to posterior primitive streak (PPS) and analyzed using scRNA-seq to investigate identity and homogeneity of the resulting population.

2022-11-30 | E-MTAB-12106 | biostudies-arrayexpress

scRNA-seq of mouse EpiSCs (epiblast stem cells) differentiated to extraembryonic mesoderm in vitro, in a monolayer

Project description:EpiSCs derived from epiblast of a gastrulating embryo differentiated towards extraembryonic mesoderm to test the efficiency of the differentiation protocol and identity and homogeneity of the resulting cell population.

2022-11-30 | E-MTAB-12107 | biostudies-arrayexpress

Single-cell RNA-seq of tailbuds from control, Pbx1/Pbx2 compound and Pbx1/Pbx2 double-knockout E8.5/E9.0 embryos.

Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). NMPs reside in the caudal lateral epiblast and tailbud, from where they sustain axial elongation. HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. To pinpoint the role of PBX proteins in the development of pre-somitic mesoderm, single-cell RNA sequencing (scRNA-seq) was performed on cells isolated from the tailbuds of control, Pbx1/Pbx2 compound (Pbx1/2-com) and Pbx1/Pbx2 double-mutant (Pbx1/2-DKO) embryos at embryonic days 8.5 and 9.0. Single cells from at least three embryos per genotype and stage were FACS-sorted into 384-well capture plates, and scRNA-seq was performed using MARS-seq (Jaitin D.A. et al., Science, 343, 776-779 (2014)). As a spike-in internal control for batch-effect correction, 71 EpiSCs cultivated in vitro were sorted into each plate, together with 311 cells from embryonic tailbuds. Two wells were left empty in each plate, as a no-cell control during data analysis.

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