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Comparative Study
. 2009 May;8(5):959-70.
doi: 10.1074/mcp.M800287-MCP200. Epub 2009 Jan 17.

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  1 , Kristoffer T G Rigbolt, Pia T Johansen, Jeanette Henningsen, Irina Kratchmarova, Moustapha Kassem, Blagoy Blagoev
Affiliations
Comparative Study

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 et al. Mol Cell Proteomics. 2009 May.

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.

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Figures

F<sc>ig</sc>. 1.
Fig. 1.
SILAC-labeled hESCs maintain self-renewal capabilities and pluripotency. A, phase-contrast images of Odense-3 hESCs cultured in normal CM or SILAC medium (top panels) and immunostaining for Oct4 self-renewal and SSEA-1 differentiation markers (middle and bottom panels, respectively). Scale bar, 100 μm. B, quantitative assessment derived from fluorescence-activated cell scanning analyses for SSEA-4 and TRA-1-81 self-renewal and SSEA-1 differentiation markers. Gray and black bars represent CM and SILAC culture conditions, respectively. Error bars represent S.D. (n = 3). C, embryoid body in vitro differentiation assay of Odense-3 hESCs grown in CM or SILAC medium showing immunostaining for markers of all three germ layers. Scale bar, 100 μm. D, reverse transcription-PCR analysis of EB and undifferentiated hESCs for self-renewal (Oct4 and Sox2), ectoderm- (NeuroD1 and MAP2), endoderm- (HNF4α, A-1-AT, and Sox17) and mesoderm (CD31)-specific markers.
F<sc>ig</sc>. 2.
Fig. 2.
SILAC labeling efficiency in human ES cells. A, overall incorporation efficiencies evaluated by assessment of SILAC ratios between unlabeled and labeled versions of 280 lysine-containing peptides (left panel) and 270 arginine-containing peptides (right panel), respectively. B, degree of arginine to proline metabolic conversion in SILAC-labeled hESCs. Arg6 was supplied in the labeling medium at a concentration 21 mg/liter (see supplemental Fig. 2 for other arginine dilutions). The extent of conversion was estimated by quantitative assessment of 107 unique proline-containing peptides.
F<sc>ig</sc>. 3.
Fig. 3.
Quantitative comparison of the proteome and phosphoproteome of SILAC-labeled hESCs and corresponding unlabeled cells grown in conventional CM. A, examples from lysine- (left panel) and arginine (right panel)-containing SILAC peptide pairs demonstrating equal expression levels of the corresponding proteins as observed by MS. Shown is the distribution of the ratios of the identified 1556 hESC proteins (B) and 527 unique phosphorylated peptides (C) presented on a log scale.
F<sc>ig</sc>. 4.
Fig. 4.
Quantitative determination of hESC membrane proteome. A, total number of membrane proteins identified from Odense-3 and HUES9 cell lines. B, distribution of protein ratios reflecting protein expression changes between differentiated and undifferentiated hESCs. Only membrane proteins identified in both Odense-3 and HUES9 cell lines with similar expression ratios (±50%) and at least one predicted transmembrane domain were included in the analysis. Ratios are averages of the values from the two cell lines and are presented on a log scale. The dashed line indicates the threshold (>3-fold change) set to discriminate potential hESC-specific markers from the remaining membrane proteins.
F<sc>ig</sc>. 5.
Fig. 5.
Protein and mRNA levels of hESC membrane markers. A, quantitative proteomics assessment: examples of SILAC peptide pairs for the corresponding proteins from differentiated (Diff.) (hollow circle) and undifferentiated (Undiff.) (solid circle) Odense-3 cells. B, mRNA levels of the selected proteins in undifferentiated and differentiated Odense-3 and HUES9 cells evaluated by real time quantitative PCR. Target gene expression levels were normalized to expression of actin; the normalized values for differentiated Odense-3 cells were then set at 1 in all panels. Error bars represent S.D. (n = 3). C, quantitative assessment on the surface expression of the six potential hESC self-renewal markers by fluorescence-activated cell scanning (see also supplemental Fig. 5). Two human ES cell lines (Odense-3 (OD3) and HUES9) were compared with somatic cells (human dermal fibroblasts (HDF)). Error bars represent S.E.M. Experiments were performed in three biological replicas.
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