Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

Tony Ly; Arlene Whigham; Rosemary Clarke; Alejandro J. Brenes-Murillo; Brett Estes; Diana Madhessian; Emma Lundberg; Patricia  Wadsworth; Angus I. Lamond

doi:10.7554/eLife.27574

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

Tony Ly (Lead / Corresponding author), Arlene Whigham, Rosemary Clarke, Alejandro J. Brenes-Murillo, Brett Estes, Diana Madhessian, Emma Lundberg, Patricia Wadsworth, Angus I. Lamond (Lead / Corresponding author)

Centre for Advanced Scientific Technologies

Research output: Contribution to journal › Article › peer-review

44 Citations (Scopus)

306 Downloads (Pure)

Abstract

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

Original language	English
Article number	e27574
Pages (from-to)	1-35
Number of pages	35
Journal	eLife
Volume	6
DOIs	https://doi.org/10.7554/eLife.27574
Publication status	Published - 20 Oct 2017

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10.7554/eLife.27574Licence: CC BY

Final Published Version
© 2017, Ly et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Final published version, 6.85 MBLicence: CC BY

Multidimensional Proteomic Analysis of Metabolic Stress & Cellular Phenotypes (Strategic Grant)
Cantrell, D. (Investigator) & Lamond, A. (Investigator)
1/01/15 → 31/12/19
Project: Research

Lamond, Angus
- Molecular Cell and Developmental Biology - Professor of Biochemistry
Person: Academic

Cite this

@article{6c945e9d248f445495350d15d7d9afd7,

title = "Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS",

abstract = "The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term {\textquoteleft}early risers{\textquoteright}. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase. ",

author = "Tony Ly and Arlene Whigham and Rosemary Clarke and Brenes-Murillo, {Alejandro J.} and Brett Estes and Diana Madhessian and Emma Lundberg and Patricia Wadsworth and Lamond, {Angus I.}",

note = "This work was supported by grants to AIL from the Wellcome Trust (105024/Z/14/Z, 108058/Z/15/Z) and the EU EpiGeneSys network (Grant#: HEALTH-F4-2010-257082). We thank the UK Research Partnership Investment Fund and the Scottish Funding Council (Project H13047) for proteomics instrumentation funding and the Wellcome Trust (097418/Z/11/Z) for supporting the Flow Cytometry and Cell Sorting Facility at the University of Dundee. We thank our colleagues in the Lamond group for advice and discussion. We thank Calum Thompson and Alan Prescott (Dundee Light Microscopy Facility), the Swedlow laboratory, and Raffaella Pippa (Lamond lab) for technical advice and assistance. ",

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doi = "10.7554/eLife.27574",

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AU - Ly, Tony

AU - Whigham, Arlene

AU - Clarke, Rosemary

AU - Brenes-Murillo, Alejandro J.

AU - Estes, Brett

AU - Madhessian, Diana

AU - Lundberg, Emma

AU - Wadsworth, Patricia

AU - Lamond, Angus I.

N1 - This work was supported by grants to AIL from the Wellcome Trust (105024/Z/14/Z, 108058/Z/15/Z) and the EU EpiGeneSys network (Grant#: HEALTH-F4-2010-257082). We thank the UK Research Partnership Investment Fund and the Scottish Funding Council (Project H13047) for proteomics instrumentation funding and the Wellcome Trust (097418/Z/11/Z) for supporting the Flow Cytometry and Cell Sorting Facility at the University of Dundee. We thank our colleagues in the Lamond group for advice and discussion. We thank Calum Thompson and Alan Prescott (Dundee Light Microscopy Facility), the Swedlow laboratory, and Raffaella Pippa (Lamond lab) for technical advice and assistance.

PY - 2017/10/20

Y1 - 2017/10/20

N2 - The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

AB - The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

U2 - 10.7554/eLife.27574

DO - 10.7554/eLife.27574

M3 - Article

C2 - 29052541

SN - 2050-084X

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SP - 1

EP - 35

JO - eLife

JF - eLife

M1 - e27574

ER -

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

Abstract

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Projects

Multidimensional Proteomic Analysis of Metabolic Stress & Cellular Phenotypes (Strategic Grant)

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Lamond, Angus

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