Dataset Information

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 (human)

TISSUE(S): Permanent Cell Line Cell, Liver, Colon

SUBMITTER: Christian Kelstrup

LAB HEAD: Jesper V. Olsen

PROVIDER: PXD004452 | Pride | 2017-06-12

REPOSITORIES: Pride

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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

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Project description:Chromatin structure and function is maintained by dynamic protein-protein and protein-nucleic acid interactions. Histones are a family of proteins that are abundant chromatin constituents and that carry numerous post-translational modifications (PTMs). Histone PTMs mediate a variety of biological activities, including recruitment of enzymatic readers, writers and erasers that modulate protein activities, DNA replication, transcription and repair. Individual histone molecules contain multiple co-existing PTMs some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. We here present an integrated experimental and computational approach for systems level molecular characterization of PTMs and PTM crosstalk. Using wildtype and engineered mouse embryonic stem cells with perturbations in the Polycomb Repressive Complex 1 (PRC1, suz12-/-), PRC2 (Ring1A/b-/-) and DNA methyltransferases (Dnmt1/3a/3b-/-) we performed comprehensive PTM analysis of histone H3 tails. We identified unique histone H3 PTM features of each of the four cell lines and we detected common combinatorial PTM features across cell lines. Using quantitative middle-down proteomics combined with probabilistic and statistical data analysis we extracted histone H3 PTM profiles for all four mESC systems. PTM crosstalk emerged as mutually exclusive histone PTMs or coordinately regulated PTMs independent of histone peptide abundance in the four model systems. We detected positive crosstalk between adjacent mono-methylated marks but strong negative crosstalk among most of the seven characterized di- and tri-methylations on lysines. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites in histone H3, including H3R2me, H3R8me and H3K37me, which exhibited specific PTM codes suggesting a particular role in chromatin. All histone H3 PTM data is available in our publicly available CrossTalkDB repository at http://crosstalkdb.bmb.sdu.dk

Dataset Information

HeLa proteome of 12,250 protein-coding genes

Publications

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

Similar Datasets

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