Proteomics Profiling of Cellular Reprogramming: Are Human Induced Pluripotent Stem Cells (hiPSCs) Indistinguishable from Human Embryonic Stem Cells (hESCs)?

<p><em>presented in: ASMS 2010 in Salt Lake City, Utah</em></p> <p> </p> <p>“Are iPS cells indistinguishable from ES cells?”<br>We prole the proteomes of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) with quantitative dimethyllabeling developed in-house to unravel the similarities and dierences between the two cell lines. Our results show that both pluripotent cell lines are nearly indistinguishable at the proteome level.</p> <p>Being self-renewing and pluripotent, hESCs hold great promise for cell transplantation therapies to cure degenerative diseases such as Parkinson’s disease or heart failures. However, this technology is limited by ethical issues since embryos are inevitably destroyed upon derivation of hESCs. Besides, potential risks do exist for immune rejection posttransplantion. These issues can probably be addressed now by the reprogramming<br>technology where adult somatic cells are induced to become hESC-like by the ectopic expression of dened transcription factors. These induced pluripotent stem cells (hiPSCs) closely resemble hESCs in many aspects, such as the expression of certain stem cell markers. More importantly, they are also capable of pluripotency and self-renewal.</p> <p>Being patient-derived and thus patient-specic, hiPSCs are ideal replacements for hESCs in cell transplantation therapies. However, before any clinical applications, they need to be characterized extensively so as to evaluate their abilities for replication and dierentiation in comparison to the actual hESCs.</p> <p>More recently, transcriptomic approaches have been used to assess the gene expression proles of both cell types. We argue that mRNA levels are not truly representative of the true character of a cell as proteins are the actual functional entities. Therefore, we used “proteomics profiling” instead and ask “Are iPS cells indistinguishable from ES cells?”</p> <p> </p> <p> </p> <p> </p

A high-resolution systems-wide screen for substrates of the SCFTrCP ubiquitin E3 ligase

<p><em>presented in: HUPO World Congress: The proteome quest to understand biology and disease in Madrid, Spain, 2013</em></p> <p> </p> <p>Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases, the largest family of enzymes in mammals. Therefore, reliable assays aimed at the identification of substrates of ubiquitin ligases are crucial, not only to unravel the molecular mechanisms by which the UPS controls protein degradation, but also for drug discovery purposes since many established UPS substrates are oncoproteins or tumor suppressors. Here, we develop a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for identifying in a systems-wide manner bone fide substrates of SCFβTrCP, a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are trademarked by a multi-subunit architecture typically comprising the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. SCFβTrCP binds, via its WD40 repeats, the DpSGXX(X)pS di-phosphorylated motif in its substrates. Our combined workflow recovers 27 previously reported SCFβTrCP substrates, of which 22 are confidently verified by two independent statistical protocols, confirming the reliability of this approach. Besides known substrates, we identify 221 proteins that, besides harboring the DpSGXX(X)pS motif, also interact specifically with the WD40 repeats. From this list, we highlight several putative novel SCFβTrCP substrates with their putative degron motifs as well as phosphorylation and ubiquitylation sites. Thus, we demonstrate that the integration of structural information, AP-MS and degron motif mining constitutes a generic, specific and effective screen for the identification of substrates of ubiquitin ligases.</p

Quantitative and qualitative liver proteome characteriztics of two inbred rat strains from integrative transcriptomics and proteomics

<p><em>presented in:Keystone Symposia: Omics Meets Cell Biology: Applications to Human Health and Disease in Taos, New Mexico, 2014</em></p> <p> </p> <p>Quantitative and qualitative protein characteristics differ between individuals and are regulated at genomic, transcriptomic and post-transcriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtained peptide evidence for 12,989 rat liver proteins, including 792 novel gene predictions, 337 novel splice events, 112 non-synonymous variants, and 54 RNA editing sites. Respectively, quantitative RNA-Seq and proteomics data correlate highly between strains, but poorly when two data types are compared within a strain, indicating extensive non-genetic regulation. Our multi-level analysis identified a genomic variant in the promoter of the most differentially expressed gene Cyp17a1, a previously reported top hit in GWAS for human hypertension, as a potential contributor to the hypertension phenotype in SHR rats. These results demonstrate the power of and need for integrative analysis for understanding genetic control of molecular dynamics and phenotypic diversity in a system-wide manner.</p