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Cell Syst. 2017 Jun 28;4(6):587-599.e4. doi: 10.1016/j.cels.2017.05.009. Epub 2017 Jun 7.

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

Author information

1
Proteomics Program, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
2
Proteomics Program, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address: christian.kelstrup@cpr.ku.dk.
3
Departments of Molecular Medicine and Clinical Medicine, Aarhus University Hospital, Aarhus University, Palle Juul-Jensens Boulevard 99, 8200 Aarhus, Denmark.
4
Institute of Pathology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus, Denmark.
5
Proteomics Program, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address: jesper.olsen@cpr.ku.dk.

Abstract

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 sequenced to a depth of ∼584,000 unique peptide sequences and ∼14,200 protein isoforms (∼12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including ∼7,000 N-acetylation sites and ∼10,000 phosphorylation sites, without the need for enrichment. We further demonstrate the general applicability and clinical potential of this proteomics strategy by comprehensively quantifying global proteome expression in several different human cancer cell lines and patient tissue samples.

KEYWORDS:

HeLa; PTM; acetylation; high pH reversed-phase fractionation; human proteome; mass spectrometry; orbitrap; patient samples; phosphorylation; proteomics

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PMID:
28601559
PMCID:
PMC5493283
DOI:
10.1016/j.cels.2017.05.009
[Indexed for MEDLINE]
Free PMC Article

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