Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Proteome Quantitation of Mouse Embryonic Stem Cells to a Depth of 5,111 Proteins

doi:10.1074/mcp.M700460-MCP200

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Proteome Quantitation of Mouse Embryonic Stem Cells to a Depth of 5,111 Proteins*S

From the Departments of ^‡Proteomics and Signal Transduction and ^‡‡Molecular Medicine, Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany and ^‖Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Roentgenstr. 20, 48149 Münster, Germany

§§To whom correspondence should be addressed. Tel.: 49-89-8578-2557; Fax: 49-89-8578-3209; E-mail: mmann{at}biochem.mpg.de

Abstract

Embryonic stem (ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are capable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC (stable isotope labeling by amino acids in cell culture)-labeled when grown feeder-free during the last phase of cell culture. We fractionated the SILAC-labeled ES cell proteome by one-dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and contains prominent stem cell markers such as OCT4, NANOG, SOX2, and UTF1 along with the embryonic form of RAS (ERAS). We also quantified the proportion of the ES cell proteome present in cytosolic, nucleoplasmic, and membrane/chromatin fractions. We compared two different preparation approaches, cell fractionation followed by one-dimensional gel separation and in-solution digestion of total cell lysate combined with isoelectric focusing, and found comparable proteome coverage with no apparent bias for any functional protein classes for either approach. Bioinformatics analysis of the ES cell proteome revealed a broad distribution of cellular functions with overrepresentation of proteins involved in proliferation. We compared the proteome with a recently published map of chromatin states of promoters in ES cells and found excellent correlation between protein expression and the presence of active and repressive chromatin marks.

Footnotes

Published, MCP Papers in Press, November 28, 2007, DOI 10.1074/mcp.M700460-MCP200
Author's Choice—Final Version Full Access.
↵¹ The abbreviations used are: ES, embryonic stem; SILAC, stable isotope labeling by amino acids in cell culture; ERAS, embryonic form of RAS; MEF, mouse embryonic fibroblast; BMP4, bone morphogenic protein 4; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; IPI, International Protein Index; GO, Gene Ontology; 1D, one-dimensional; GeLCMS, in-gel digest followed by LC-MS/MS; ID, identity; ChIPseq, chromatin immunoprecipitation together with large scale sequencing of the occupied DNA region; H3K4me3, histone 3 lysine 4 trimethylation, H3K27me3, histone 3 lysine 27 trimethylation; P, present.
↵* This work was supported in part by the European Union Grant High-throughput Epigenetic Regulatory Organisation In Chromatin (HEROIC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
↵§ Both authors contributed equally to this work.
↵¶ Supported by the European Network Grant RUBICON.
↵** Supported by the Interdepartmental Graduate Program for Experimental Life Sciences (IGEL) program (University of Münster).
- Received September 25, 2007.
- Revision received November 19, 2007.