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

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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.

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Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

Shao-En Ong, Blagoy Blagoev, Irina Kratchmarova … (2002)

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

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MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes

Graeme C McAlister, David Nusinow, Mark P. Jedrychowski … (2014)

Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8 378 proteins in union and 6 168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174 704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.

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The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit.

Sabrina L Spencer, Steven D Cappell, Feng-Chiao Tsai … (2013)

Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity. Copyright © 2013 Elsevier Inc. All rights reserved.

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Author and article information

Contributors

Tony Ly:

ORCID: https://orcid.org/0000-0002-8650-5215

Arlene Whigham

Rosemary Clarke

Alejandro J Brenes-Murillo

Brett Estes

Diana Madhessian

Emma Lundberg

Patricia Wadsworth

Angus I Lamond:

ORCID: http://orcid.org/0000-0001-6204-6045

Jon Pines: Role: Reviewing Editor

Journal

Journal ID (nlm-ta): eLife

Journal ID (iso-abbrev): Elife

Journal ID (publisher-id): eLife

Title: eLife

Publisher: eLife Sciences Publications, Ltd

ISSN (Electronic): 2050-084X

Publication date (Electronic, pub): 20 October 2017

Publication date Collection: 2017

Volume: 6

Electronic Location Identifier: e27574

Affiliations

[1 ]deptCentre for Gene Regulation and Expression, School of Life Sciences University of Dundee DundeeUnited Kingdom

[2 ]deptWellcome Centre for Cell Biology University of Edinburgh EdinburghUnited Kingdom

[3 ]deptCAST Flow Cytometry Facility, School of Life Sciences University of Dundee DundeeUnited Kingdom

[4 ]deptDepartment of Biology University of Massachusetts MassachusettsUnited States

[5 ]deptProgram in Molecular and Cellular Biology University of Massachusetts MassachusettsUnited States

[6 ]deptScience for Life Laboratory Royal Institute of Technology StockholmSweden

The Gurdon Institute United Kingdom

Author information

Tony Ly https://orcid.org/0000-0002-8650-5215

Angus I Lamond http://orcid.org/0000-0001-6204-6045

Article

Publisher ID: 27574

DOI: 10.7554/eLife.27574

PMC ID: 5650473

PubMed ID: 29052541

SO-VID: 85a94310-b13c-4970-8e3c-1322a0de599f

License:

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.

History

Date received : 11 April 2017

Date accepted : 06 October 2017

Funding

Funded by: Knut och Alice Wallenbergs Stiftelse;

Award Recipient : Emma Lundberg

Funded by: FundRef http://dx.doi.org/10.13039/100010269, Wellcome;

Award ID: 105024/Z/14/Z