Quantitative Proteome Analysis of Pluripotent Cells by iTRAQ Mass Tagging Reveals Post-transcriptional Regulation of Proteins Required for ES Cell Self-renewal

Abstract

Embryonic stem cells and embryonal carcinoma cells share two key characteristics: pluripotency (the ability to differentiate into endoderm, ectoderm, and mesoderm) and self-renewal (the ability to grow without change in an untransformed, euploid state). Much has been done to identify and characterize transcription factors that are necessary or sufficient to maintain these characteristics. Oct-4 and Nanog are necessary to maintain pluripotency; they are down-regulated at the mRNA level by differentiation. There may be additional regulatory genes whose mRNA levels are unchanged but whose proteins are destabilized during differentiation. We generated proteome-wide, quantitative profiles of ES and embryonal carcinoma cells during differentiation, replicating a microarray-based study by Aiba et al. We identified several proteins whose levels decreased during differentiation in both cell types but whose mRNA levels were unchanged. We confirmed several of these cases by RT-PCR and Western blot. Racgap1 (also known as mgcRacgap) was particularly interesting because it is required for viability of preimplantation embryos and hematopoietic stem cells, and it is also required for differentiation. To confirm our observation that RACGAP-1 declines during retinoic acid-mediated differentiation, we used multiple reaction monitoring, a targeted mass spectrometry-based quantitation method, and determined that RACGAP-1 levels decline by half during retinoic acid-mediated differentiation. We knocked down Racgap-1 mRNA levels using a panel of five shRNAs. This resulted in a loss of self-renewal that correlated with the level of knockdown. We conclude that RACGAP-1 is post-transcriptionally regulated during blastocyst development to enable differentiation by inhibiting ES cell self-renewal. Molecular & Cellular Proteomics 9: 2238 -2251, 2010.

Quantitative Proteomic and Functional Analysis of Liver Mitochondria from High Fat Diet (HFD) Diabetic Mice

“…Our studies failed to show fatty acid metabolism, pyruvate metabolism and TCA cycle that have been shown to be up-regulated in previous studies (18,19). Our experience is consistent with the findings from O'Brien et al (31) that the t test was not an adequate statistical measure of the reproducibility of protein changes using iTRAQ. The reason might be that isobaric tags FIG.…”

Upregulation of Rac GTPase-Activating Protein 1 Is Significantly Associated with the Early Recurrence of Human Hepatocellular Carcinoma

“…5C). This is likely to be associated with the differentiation of RACGAP1 depleted cells as suggested by O'Brien and colleagues (22).…”

Clinical significance of RacGAP1 expression at the invasive front of gastric cancer

“…We think a reason for the difference could be that the IHC score was calculated by evaluation of whole tumor, while the tissue samples for the evaluation of mRNA level were obtained from part of the tumor. O 0 Brien et al [25] demonstrated that RacGAP1 was required for differentiation of embryos and hematopoietic stem cells and concluded that RACGAP1 was post-transcriptionally regulated during blastocyte development to enable embryonic stem cell self-renewal. These mechanisms may explain the apparent differences of RacGAP1 expression determined by immunohistochemical and transcriptional analyses.…”