Rong Lu et al. present a dynamic systems-level study of cell fate changes in mouse embryonic stem cells following a single well-defined perturbation by downregulating the pluripotency factor Nanog. They measure global changes in histone acetylation, chromatin-bound RNA polymerase II, mRNA and nuclear protein levels for five days, and find progressive widespread changes in multiple molecular regulatory layers and provide a dynamic view of information flow in the epigenome, transcriptome and proteome. The interaction between the different molecular mechanisms that regulate changes in embryonic stem cell (ESC) fate is not fully understood. A dynamic systems-level study of cell fate change in murine ESCs following a well-defined perturbation is now presented; the data demonstrate how a single genetic perturbation leads to widespread changes in several molecular regulatory layers, and provide a dynamic view of information flow in the epigenome, transcriptome and proteome. Molecular regulation of embryonic stem cell (ESC) fate involves a coordinated interaction between epigenetic1,2,3,4, transcriptional5,6,7,8,9,10 and translational11,12 mechanisms. It is unclear how these different molecular regulatory mechanisms interact to regulate changes in stem cell fate. Here we present a dynamic systems-level study of cell fate change in murine ESCs following a well-defined perturbation. Global changes in histone acetylation, chromatin-bound RNA polymerase II, messenger RNA (mRNA), and nuclear protein levels were measured over 5 days after downregulation of Nanog, a key pluripotency regulator13,14,15. Our data demonstrate how a single genetic perturbation leads to progressive widespread changes in several molecular regulatory layers, and provide a dynamic view of information flow in the epigenome, transcriptome and proteome. We observe that a large proportion of changes in nuclear protein levels are not accompanied by concordant changes in the expression of corresponding mRNAs, indicating important roles for translational and post-translational regulation of ESC fate. Gene-ontology analysis across different molecular layers indicates that although chromatin reconfiguration is important for altering cell fate, it is preceded by transcription-factor-mediated regulatory events. The temporal order of gene expression alterations shows the order of the regulatory network reconfiguration and offers further insight into the gene regulatory network. Our studies extend the conventional systems biology approach to include many molecular species, regulatory layers and temporal series, and underscore the complexity of the multilayer regulatory mechanisms responsible for changes in protein expression that determine stem cell fate.

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRβ, FIP1/PDGFRα, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRα. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that currently affects 36 million people worldwide with no effective treatment available. Development of AD follows a distinctive pattern in the brain and is poorly modelled in animals. Therefore, it is vital to widen the spatial scope of the study of AD and prioritise the study of human brains. Here we show that functionally distinct human brain regions display varying and region-specific changes in protein expression. These changes provide insights into the progression of disease, novel AD-related pathways, the presence of a gradient of protein expression change from less to more affected regions and a possibly protective protein expression profile in the cerebellum. This spatial proteomics analysis provides a framework which can underpin current research and open new avenues to enhance molecular understanding of AD pathophysiology, provide new targets for intervention and broaden the conceptual frameworks for future AD research.

Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.