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

• 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.

Subject headings

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Keyword

membrane protein

rrm2 protein

tpx2 protein

unclassified drug

analytic method

Article

cell cycle progression

controlled study

flow cytometry

fluorescence activated cell sorting

gene overexpression

high performance liquid chromatography

human

human cell

immunofluorescence microscopy

immunofluorescence test

interphase

liquid chromatography-mass spectrometry

mass spectrometry

mitosis

protein expression

protein phosphorylation

PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets

proteomics

Western blotting

Publication and Content Type

ref (subject category)

art (subject category)