Flagging False Positives Following Untargeted LC-MS Characterization of Histone Post-Translational Modification Combinations.

Epigenetic changes can be studied with an untargeted characterization of histone post-translational modifications (PTMs) by liquid chromatography-mass spectrometry (LC-MS). While prior information about more than 20 types of histone PTMs exists, little is known about histone PTM combinations (PTMCs). Because of the combinatorial explosion it is intrinsically impossible to consider all potential PTMCs in a database search.

Extracting histones for the specific purpose of label-free MS.

Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS.

Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells.

We present a fully defined culture system (adapted Essential8 [E8 ] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8 medium was modified by adding (1) l-proline, (2) l-ornithine, (3) N -hydroxy-nor-l-arginine acetate, or by (4) lowering the arginine concentration.

Tackling aspecific side reactions during histone propionylation: The promise of reversing overpropionylation.

Histone proteins are essential elements for DNA packaging. Moreover, the PTMs that are extremely abundant on these proteins, contribute in modeling chromatin structure and recruiting enzymes involved in gene regulation, DNA repair and chromosome condensation. This fundamental aspect, together with the epigenetic inheritance of histone PTMs, underlines the importance of having biochemical techniques for their characterization.

Phospho-iTRAQ data article: Assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.

The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part (Pflieger et al., 2008.

Pitfalls in histone propionylation during bottom-up mass spectrometry analysis.

Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom-up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation.

Phospho-iTRAQ: assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.

The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Our use of isobaric tags for relative and absolute quantitation (iTRAQ) marks the first time that isobaric tags have been applied for the large-scale analysis of phosphopeptides.

Histone proteolysis: a proposal for categorization into 'clipping' and 'degradation'.

We propose for the first time to divide histone proteolysis into "histone degradation" and the epigenetically connoted "histone clipping". Our initial observation is that these two different classes are very hard to distinguish both experimentally and biologically, because they can both be mediated by the same enzymes. Since the first report decades ago, proteolysis has been found in a broad spectrum of eukaryotic organisms.

Detailed method description for noninvasive monitoring of differentiation status of human embryonic stem cells.

The (non)differentiation status of human embryonic stem cells (hESCs) is usually analyzed by determination of key pluripotency defining markers (e.g., OCT4, Nanog, SOX2) by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry (FC), and immunostaining.

Identification of histone H3 clipping activity in human embryonic stem cells.

Posttranslational histone modifications are essential features in epigenetic regulatory networks. One of these modifications has remained largely understudied: regulated histone proteolysis. In analogy to the histone H3 clipping during early mouse embryonic stem cell differentiation, we report for the first time that also in human embryonic stem cells this phenomenon takes place in the two different analyzed cell lines.