Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

Tatyana A Prokhorova, Kristoffer T G Rigbolt, Pia T Johansen, Jeanette Henningsen, Irina Kratchmarova, Moustapha Kassem, Blagoy Blagoev

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.
Original languageEnglish
JournalMolecular and Cellular Proteomics
Volume8
Issue number5
Pages (from-to)959-70
Number of pages11
ISSN1535-9476
DOIs
Publication statusPublished - 1. May 2009

Keywords

  • Amino Acids
  • Animals
  • Biological Markers
  • Cell Differentiation
  • Cell Proliferation
  • Cells, Cultured
  • Culture Media, Conditioned
  • Embryonic Stem Cells
  • Gene Expression Regulation, Developmental
  • Humans
  • Isotope Labeling
  • Membrane Proteins
  • Mice
  • Phosphoproteins
  • Pluripotent Stem Cells
  • Proteome
  • Proteomics
  • RNA, Messenger

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