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Elife. 2017 Oct 20;6. pii: e27574. doi: 10.7554/eLife.27574.

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

Author information

1
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
2
Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
3
CAST Flow Cytometry Facility, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
4
Department of Biology, University of Massachusetts, Massachusetts, United States.
5
Program in Molecular and Cellular Biology, University of Massachusetts, Massachusetts, United States.
6
Science for Life Laboratory, Royal Institute of Technology, Stockholm, Sweden.

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, 2015). Here, we show that by using specific intracellular immunolabelling 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 iMMUnolabelled 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, termed '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 the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

KEYWORDS:

FACS; biochemistry; cell biology; cell cycle; human; mitosis; phosphoproteomics; proteomics

PMID:
29052541
PMCID:
PMC5650473
DOI:
10.7554/eLife.27574
[Indexed for MEDLINE]
Free PMC Article

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