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

TY - JOUR

T1 - An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes

AU - Bekker-Jensen, Dorte B

AU - Kelstrup, Christian D

AU - Batth, Tanveer S

AU - Larsen, Sara C

AU - Haldrup, Christa

AU - Bramsen, Jesper B

AU - Sørensen, Karina D

AU - Høyer, Søren

AU - Ørntoft, Torben F

AU - Andersen, Claus L

AU - Nielsen, Michael L

AU - Olsen, Jesper V

N1 - Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

PY - 2017/6/28

Y1 - 2017/6/28

N2 - 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.

AB - 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.

KW - Journal Article

U2 - 10.1016/j.cels.2017.05.009

DO - 10.1016/j.cels.2017.05.009

M3 - Journal article

C2 - 28601559

SN - 2405-4712

VL - 4

SP - 587-599, e1-e4

JO - Cell Systems

JF - Cell Systems

IS - 6

ER -