Proteomic analysis of stem cell differentiation and early development

Abstract

Genomics methodologies have advanced to the extent that it is now possible to interrogate the gene expression in a single cell but proteomics has traditionally lagged behind and required much greater cellular input and was not quantitative. Coupling protein with gene expression data is essential for understanding how cell behavior is regulated. Advances primarily in mass spectrometry have, however, greatly improved the sensitivity of proteomics methods over the last decade and the outcome of proteomic analyses can now also be quantified. Nevertheless, it is still difficult to obtain sufficient tissue from staged mammalian embryos to combine proteomic and genomic analyses. Recent developments in pluripotent stem cell biology have in part addressed this issue by providing surrogate scalable cell systems in which early developmental events can be modeled. Here we present an overview of current proteomics methodologies and the kind of information this can provide on the biology of human and mouse pluripotent stem cells.

Figure 1.

Workflow for the proteomic analysis of biological samples, from sample preparation to biological validation. The indicated techniques are a nonexhaustive list of examples, which often are used in combination.

Figure 2.

Western blots showing wild-type SOX2 and SOX2 mutants expressed in HeLa cells. HeLa cells that were transfected with 6 × histidine-tagged SUMO2 in combination with either wild-type SOX2 (lane 2) or SOX2 mutants where serine residues 249, 250, and 251 were all (lane 3) or individually (lanes 5–7) mutated to aspartic acid residues, which mimics constitutive phosphorylation of the respective serine residues. SUMO2-SOX2 complexes were purified from the cell lysates with Ni-NTA beads binding the 6 × histidine tag of SUMO2, and then subjected to SDS-PAGE and Western blotting using a SOX2-specific antibody. Wild-type SOX2 (lane 2) is marginally SUMOylated; mutating lysine 245 into alanine (lane 4) shows that this residue is the SUMO target site. Whereas mutation of serine 250 into aspartic acid (lane 6) has no pronounced effect, mutating either all three serine residues simultaneously (lane 3) or serine 249 (lane 5) or 251 (lane 7) individually results in increased polySUMOylation. Mock: negative control of HeLa cells transfected with an empty plasmid (D Van Hoof, J Krijgsveld, and CL Mummery, unpubl.).

Figure 3.

Schematic of the effects of phosphorylation and subsequent SUMOylation of SOX2 in self-renewing and differentiating pluripotent stem cells. The results from the study by Rigbolt et al. (2011) and our own data (Van Hoof et al. 2009; and the unpublished results of Fig. 1) imply that there is a fine balance between the transcription/translation and degradation rates of SOX2 to sustain self-renewal (A). If the transcription/translation feedback loop is disturbed by, e.g., PMA, the continuous phosphorylation, the subsequent SUMOylation, and the eventual degradation of the remaining SOX2 proteins lead to a steady decrease in SOX2 levels, resulting in a shift toward differentiation (B). On the other hand, if the intracellular levels of SOX2 increase owing to a decrease in the degradation rate, the cells will also differentiate as a result of the transcription/translation of differentiation-associated genes (C). The dotted lines indicate that the precise action and targets of these differentiation-inducing conditions are not known. Abbreviations: PMA, phorbol 12-myristate 13-acetate; NCM, non-conditioned medium.