Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics

ABSTRACT: This project used a custom-built capillary electrophoresis (CE) mass spectrometry (MS) platform to demonstrate the scalable analysis of proteins from single cells of varying sizes and protein content in different vertebrate biological models. Neural tissue fated giant cells were identified in cleavage-stage Xenopus laevis (frog) embryos, and 18 ng from its protein content was analyzed. From cultured primary neurons from the mouse, diluted samples containing ~125 pg of protein digest was measured, estimating to a portion of the somal protein content. Compared to traditional nano-flow liquid chromatography (nanoLC) MS, CE-MS provided higher sensitivity and faster analysis. CE-ESI-HRMS offers a viable approach for scalable single-cell proteomics.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Xenopus Laevis (african Clawed Frog) Mus Musculus (mouse)

TISSUE(S): Brain, Embryo, Early Embryonic Cell

SUBMITTER: Peter Nemes

LAB HEAD: Peter Nemes

PROVIDER: PXD030607 | Pride | 2023-10-25

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	2016-04-01_SC03_hip_cultured_neu_0.5_ugul.RAW	Raw
	2016-04-04_SC02_hip_cultured_neu_0.5_ugul.RAW	Raw
	2016-04-04_SC02_hip_cultured_neu_lower_ce_hv_20kv_wit_mass_excl_sc01.msf	Msf
	2016-04-06_SC02_hip_cultured_neu_lower_ce_hv_20kv.msf	Msf
	2016-04-11_SC04_hip_cultured_neuron_0.125_ugul.RAW	Raw

Items per page:

1 - 5 of 37

Similar Datasets

Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics

Project description:This project used a custom-built capillary electrophoresis (CE) mass spectrometry (MS) platform to demonstrate the scalable analysis of proteins from single cells of varying sizes and protein content in different vertebrate biological models. Neural tissue fated giant cells were identified in cleavage-stage Xenopus laevis (frog) embryos, and 18 ng from its protein content was analyzed. From cultured primary neurons from the mouse, diluted samples containing ~125 pg of protein digest was measured, estimating to a portion of the somal protein content. Compared to traditional nano-flow liquid chromatography (nanoLC) MS, CE-MS provided higher sensitivity and faster analysis. CE-ESI-HRMS offers a viable approach for scalable single-cell proteomics.

2022-03-02 | PXD031955 | Pride

The 15-min Subcellular Proteome

Project description:This project integrated data-independent acquisition (DIA) with capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) to enable fast single-cell proteomics. With <15 min of effective separation time in a bottom-up proteomics setting, this technology returned ~1,200 proteins from a single HeLa-cell-equivalent proteome amount. Furthermore, using capillary microsampling to collect the proteome from identified cells in a cleavage-stage embryo of the vertebrate Xenopus laevis (frog) embryo, ~1,400 proteins were identified by measuring ~5 ng of the subcellular proteome content aspirated from live Xenopus embryos. CE-ESI-MS with DIA enhanced proteome detection sensitivity and improved analytical throughput.

2024-09-27 | PXD046467 | Pride

H3 N-terminal tail peptide interactome

Project description:The closely related replicative H3 and non-replicative H3.3 variants show specific requirement during development in vertebrates. Whether it involves distinct mode of deposition or unique roles once incorporated into chromatin remains unclear. To disentangle the two aspects, we took advantage of the Xenopus early development combined with chromatin assays. We systematically mutated H3.3 at each four residues that differ from H3.2 and tested their ability to rescue developmental defects due to endogenous H3.3 depletion. Surprisingly, all H3.3 mutated variants functionally complemented endogenous H3.3, regardless of their incorporation pathways, except for one residue, the serine at position 31. The phosphorylation at this unique residue occurs onto chromatin with a peak in late mitosis, and depends on the networks of cell cycle kinases. Notably, while the alanine substitution failed to rescue H3.3 depletion, a phosphomimic residue sufficed. Based on proteomics studies with histone peptides and Xenopus extracts, we find that the phosphomimic histone mutant attracts transcription related factors. Furthermore, we evidence a crosstalk whereby phosphorylation on H3.3S31 favors H3.3K27ac. At gastrulation, we conclude that the critical importance of the H3.3S31 residue is independent of the variant incorporation pathway. It rather reflects a signaling role engaging key binding partners and crosstalks on neighboring amino acids. We discuss how this single evolutionary conserved residue conveys both in interphase and mitosis unique properties for this variant in vertebrates during cell cycle and cell fate commitment.

2020-03-13 | PXD016497 | Pride

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

Project description:In this project, we enabled mass spectrometry based proteomics characterization of single neurons in sections of the mouse brain. We accessed cells in a whole-cell patch clamp configuration and aspirated a portion of the neuronal soma into the patch clamp probe. The collected aspirate was expelled into a microvial, where proteins were extracted and processed for shot-gun analysis. The resulting trace amounts of protein digests were analyzed on a custom-built microanalytical capillary electrophoresis platform that was connected to an electrospray ionization high-resolution mass spectrometer. This “patch proteomics” technology identified ~150 proteins from triplicate measurement of single dopaminergic neurons in the mouse substantia nigra.

2022-02-18 | PXD028040 | Pride

The identification of differentially expressed genes between animal and vegetal blastomeres in Xenopus laevis

Project description:To identify asymmetrically localized maternal mRNAs along the animal-vegetal axis in cleavage Xenopus embryos, we isolated animal and vegetal blastomeres at 8-cell stage, extracted the maternal mRNA respectively and analyzed them by RNA-seq technology. We identified 43 maternal transcripts significantly enriched in the vegetal region (FDR<0.05) by R/Bioconductor package DESeq RNAseq of animal and vegetal blastomeres with 2 biological replicates

2015-05-19 | E-GEOD-68972 | biostudies-arrayexpress

Expression data from juvenile Xenopus laevis inner ear tissue

Project description:We implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. Gene expression analysis of Xenopus laevis juvenile inner ear tissue. Inner ear RNA isolated from three groups of 5-10 juvenile X. laevis. Each biological replicate represents pooled inner ear RNA from 10-19 inner ears.

2012-05-04 | E-GEOD-37767 | biostudies-arrayexpress

The translation regulator Zar1l is required to control the timing of meiosis in Xenopus oocytes

Project description:In vertebrates, sexual reproduction depends on the precisely temporally controlled translation of mRNAs stockpiled in the oocyte. The RNA-binding proteins Zar1 and Zar2 have been implicated in translational control in oocytes of several vertebrate species, but how they act mechanistically is still incompletely understood. Here, we investigate the function of Zar1l, a so far uncharacterized member of the Zar protein family, in Xenopus laevis oocytes. By combining biochemical assays and mass spectrometry, we reveal that Zar1l is a constituent of a known large ribonucleoparticle containing the translation repressor 4E-T and the central polyadenylation regulator CPEB1. Employing TRIM-Away, we show that depletion of Zar1l from prophase-I arrested oocytes results in premature meiotic maturation upon hormone treatment. We provide evidence that this is based on the precocious expression of the kinase cMos, a key promotor of meiotic resumption. Based on our data, we propose a model according to which degradation of Zar1l results in dissociation of 4E-T from CPEB1, thus weakening translation inhibition imposed by the mRNA 3’UTR of cMos and probably also other M-phase promoting regulators. Thus, our detailed characterization of the function of Zar1l reveals novel mechanistic insights into the regulation of maternal mRNA translation during vertebrate meiosis.

2022-09-22 | PXD032170 | Pride

Regulation of mRNA localization during the oocyte-to-embryo transition by regulated mRNA-endoplasmic reticulum association and remodeling of the endoplasmic reticulum

Project description:As an important post-transcriptional regulatory mechanism, asymmetric localization of mRNAs is essential for cell polarity and cell fate determination during early development. In an effort to understand localization of mRNAs encoding proteasome components during Xenopus oocyte-to-embryo transition, we discovered that the endoplasmic reticulum (ER), which is thought to mainly serve “house-keeping” roles in the cell, plays a multifaceted role in controlling localization of maternal RNAs. Our RNA-seq analysis of fractionated transcripts reveals that more than 40% of RNAs, including proteasome mRNAs, are present on the ER in Xenopus oocytes. After meiotic maturation, a large fraction of ER-associated RNA is released into the cytosol. The release of proteasome RNAs from the ER into the cytosol is conserved during mouse oocyte maturation. Our comparative proteomic analysis and ribonucleoprotein immunoprecipitation demonstrate that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation, providing a mechanistic explanation for the dynamic regulation of RNA-ER association. We further show that the ER is remodeled massively during Xenopus oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of proteasome mRNAs in mature eggs. To our knowledge, our findings demonstrate for the first time that dynamic regulation of RNA-ER association and remodeling of the ER are fundamentally important for asymmetric localization of RNAs during development.

2022-12-15 | PXD033018 | Pride

Fe3+-NTA magnetic beads as an alternative to spin column-based phosphopeptide enrichment

Project description:Protein phosphorylation is a central mechanism of cellular signal transduction in living organisms. Phosphoproteomic studies aim to systematically catalogue, characterize, and comprehend alterations in phosphorylation states across multiple cellular conditions and are often incorporated into global proteomics workflows. Previously, we found that spin column-based Fe3+-NTA enrichment integrated well with our workflow but it remained a bottleneck for methods that require higher throughput or a scale that is beyond the maximum capacity of these columns. Here, we compare our well-established spin column-based enrichment strategy with one encompassing magnetic beads. Our data show little difference in both the number and properties of the phosphopeptides identified when using either method. In all, we illustrate how scalable and automation-friendly magnetic Fe3+-NTA beads can seamlessly substitute spin column-based Fe3+-NTA for global phosphoproteome profiling.

2022-09-02 | PXD031327 | Pride

hnRNP R Knock-out affects the axonal and somatic proteome

Project description:We grew neurons in microchambers to separate axons and soma in different compartments which could be independently harvested for proteomic analysis. Using this model system, we studied the effect of the knock-out of hnRNP R on the axonal and somatic proteome.

2024-08-22 | PXD038368 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data