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HeLa proteome of 12,250 protein-coding genes

ABSTRACT: We developed an optimized multi-shot proteomics workflow based on high-resolution offline high pH reversed-phase peptide separation of high peptide loads collecting many fractions that were in turn analyzed by short online chromatographic separations and fast peptide sequencing using orbitrap tandem mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Jesper V. Olsen

PROVIDER: MSV000081563 | MassIVE |

SECONDARY ACCESSION(S): PXD004452

REPOSITORIES: MassIVE

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Publications

An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes.

Bekker-Jensen Dorte B DB Kelstrup Christian D CD Batth Tanveer S TS Larsen Sara C SC Haldrup Christa C Bramsen Jesper B JB Sørensen Karina D KD Høyer Søren S Ørntoft Torben F TF Andersen Claus L CL Nielsen Michael L ML Olsen Jesper V JV

Cell systems 20170607 6

This study investigates the challenge of comprehensively cataloging the complete human proteome from a single-cell type using mass spectrometry (MS)-based shotgun proteomics. We modify a classical two-dimensional high-resolution reversed-phase peptide fractionation scheme and optimize a protocol that provides sufficient peak capacity to saturate the sequencing speed of modern MS instruments. This strategy enables the deepest proteome of a human single-cell type to date, with the HeLa proteome se ...[more]

PMID: 28601559

Similar Datasets

HeLa proteome of 12,250 protein-coding genes

Project description:We developed an optimized multi-shot proteomics workflow based on high-resolution offline high pH reversed-phase peptide separation of high peptide loads collecting many fractions that were in turn analyzed by short online chromatographic separations and fast peptide sequencing using orbitrap tandem mass spectrometry.

2017-06-12 | PXD004452 | Pride

Rapid and Deep Proteomes by Faster Sequencing on a Benchtop Quadrupole Ultra-High-Field Orbitrap Mass Spectrometer

Project description:The faster sequencing speed available on the latest Q Exactive HF mass spectrometer is investigated and evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments. Also the instrument capabilities for offline high pH reversed-phase peptide fractionation and single-shot phosphoproteomics are evaluated.

2014-10-31 | PXD001305 | Pride

Rapid and Deep Proteomes by Faster Sequencing on a Benchtop Quadrupole Ultra-High-Field Orbitrap Mass Spectrometer

2016-03-01 | MSV000079548 | MassIVE

Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing

Project description:In large-scale quantitative mass spectrometry (MS)-based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high-pH reversed-phase peptide chromatographic fractionation maximizes depth of coverage. To investigate to what extent limited sample amounts affects sensitivity and dynamic range of the analysis due to sample losses, we benchmarked TMT-based peptide fractionation strategies against single-shot (SS) label-free approach with data independent acquisition (DIA), for different peptide input per sample. To systematically examine how peptide input amounts influence TMT-fractionation approaches in a phosphoproteomics workflow, we compared two different high-pH reverse-phase fractionation strategies, microflow (MF) and stage-tip fractionation (STF), while scaling the peptide input amount down from 12.5 μg to 1 μg per sample. Our results indicate that, for input amounts higher than 5 μg per sample, TMT labeling, followed by microflow fractionation and phospho-enrichment (MF), achieves the deepest phosphoproteome coverage, even compared to SS DIA analysis. Conversely, stage-tip fractionation of enriched phosphopeptides (STF) is optimal for lower amounts, below 5 μg/peptide per sample. As a result, we provide a decision tree to help phosphoproteomics users to choose the best workflow as a function of on sample amount.

2022-06-28 | PXD031277 | Pride

Dual targeted extracellular vesicles regulating oncogenic genes in pancreatic cancer [sRNAseq]

Project description:Pancreatic ductal adenocarcinoma (PDAC) tumors carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to high solid tumor burden like PDAC. Here, we report a dual-targeted extracellular vesicle (EV) carrying high loads of RNA that effectively suppresses large PDAC tumors in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation released abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dual-targeted EVs effectively suppressed large orthotopic PANC-1 and patient derived xenograft tumors in mice and extended animal survival. This work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumors.

2023-10-02 | GSE223397 | GEO

Dual targeted extracellular vesicles regulating oncogenic genes in pancreatic cancer [Mouse Array]

Project description:Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to high solid tumour burden like PDAC. Here, we report a dual-targeted extracellular vesicle (EV) carrying high loads of RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation released abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dual-targeted EVs effectively suppressed large orthotopic PANC-1 and patient derived xenograft tumours in mice and extended animal survival. This work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.

2023-10-02 | GSE223408 | GEO

Dual targeted extracellular vesicles regulating oncogenic genes in pancreatic cancer [Human Array]

2023-10-02 | GSE223407 | GEO

High contaminant loads in Lake Apopka mesocosms affect the ovarian transcriptome in largemouth bass

Project description:This SuperSeries is composed of the following subset Series: GSE38738: High contaminant loads in Lake Apopka mesocosms affect the ovarian transcriptome in largemouth bass [April] GSE38773: High contaminant loads in Lake Apopka mesocosms affect the ovarian transcriptome in largemouth bass [January] Refer to individual Series

2012-08-31 | E-GEOD-38774 | biostudies-arrayexpress

Insulin-like 3 affects zebrafish spermatogenic cells directly and via Sertoli cells

Project description:Testis tissue was incubated under basal conditions for 2 days to allow for the assumed decrease of endogenous Insl3 production, then added exogenous, recombinant zebrafish Insl3 peptide (100 ng/mL) and continued the incubation for another 2 days, before collecting the tissue for analysis of gene expression

2021-03-02 | GSE152038 | GEO

Trimodal chromatography enables facile peptide fractionation

Project description:We evaluated the utility of a trimodal stationary phase (Trinity P1) in offline peptide fractionation, and found it performed similarly to ion exchange or high pH reversed phase based techniques. It is orthogonal to low pH reversed phase and uses MS compatible mobile phase solvents.

2017-08-07 | PXD006188 | Pride

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