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.
Highlights
The mitotic cell division cycle is composed of four major phases, that is, G1, S, G2 and M
We identified three steps in intracellular immunostaining procedures that have the potential to significantly impact peptide identification by mass spectrometry (MS)-based proteomics: (1) irreversibility and/or chemical modifications associated with cell fixation, (2) loss of soluble proteins during permeabalisation and (3) interference from antibody-derived peptides (Figure 1A)
We characterise the proteomics of intracellular immunolabelled cell subsets (PRIMMUS) method, which provides a flexible approach that facilitates quantitative proteomic studies on specific cell subsets isolated by Fluorescence Activated Cell Sorting (FACS) based on staining for intracellular antigens
Summary
The mitotic cell division cycle is composed of four major phases, that is, G1, S, G2 and M. The phases are defined by two major events during cell division: DNA replication (S phase) and mitosis (M phase), with intervening gap phases (G1 and G2). The cell cycle is driven by the expression of key proteins, called cyclins. Cyclin expression and function is restricted to specific cell cycle phases, driving temporally ordered phosphorylation of key substrates by interacting with their kinase partners, the cyclin-dependent kinases (CDKs). Regulated degradation of the cyclins ensures that progression through the cell cycle is unidirectional. Cyclin A is targeted for degradation by the anaphase promoting complex/cyclosome (APC/C), a multiprotein E3 ubiquitin ligase, restricting cyclin A-driven phosphorylation to S, G2 and early M-phase
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