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. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom-up MS on histones

PiTRAQ, a strategy to simultaneously correlate protein expression and phosphorylation stoichiometry between different samples: evaluation on different mass spectrometers

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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. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885

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. Over the past two decades, significant improvements in mass accuracy and resolution of mass spectrometers have made LC-coupled MS the strategy of choice for accurate identification and quantification of protein PTMs. Nevertheless, in previous work we disclosed the limitations and biases of the most widely adopted sample preparation protocols for histone propionylation, required prior to bottom-up MS analysis. In this work, however, we put forward a new specific and efficient propionylation strategy by means of propionic anhydride. In this method, aspecific overpropionylation at serine (S), threonine (T) and tyrosine (Y) is reversed by adding hydroxylamine (HA). We recommend using this method for future analysis of histones through bottom-up MS

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. Despite proven usefulness of these techniques, their destructive nature makes it impossible to follow up on the same hESC colonies for several days, leading to a loss of information. In 2003, an OCT4-eGFP knock-in hESC line to monitor OCT4 expression was developed and commercialized. However, to the best of our knowledge, the use of fluorescence microscopy (FM) for monitoring the OCT4-eGFP expression of these cells without sacrificing them has not been described to date. Here, we describe such a method in detail, emphasizing both its resolving power and its complementary nature to FC as well as the potential pitfalls in standardizing the output of the FM measurements. The potential of the method is demonstrated by comparison of hESCs cultured in several conditions, both feeder free (vitronectin, VN) and grown on feeder cells (mouse embryonic fibroblasts, MEFs)

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. Mol. Cell. Proteomics, 7: 326–46 [1]; Wu et al., 2011. Nat. Methods, 8: 677–83 [2]). Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of PhosphoiTRAQ with regards to the experimental setup and data mining, the proteome coverage was extended through gel fractionation, while an internal replicate measurement creates more stringent data analysis opportunities. The latter allows other researchers to set their own threshold for selecting potential phosphorylation events in the dataset presented here, depending on the biological question or corroboration under investigation. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data accompanying the manuscript on this approach (Glibert et al., 2015, J. Proteome Res. 14: 2015, 839–49 [5]) have been deposited to the ProteomeXchange with identifier PXD001574

Flagging false positives following untargeted LCMS characterization of histone PTM combinations

Epigenetic changes can be studied with an untargeted characterization of histone post-translational modifications (PTMs) by LCMS. While prior information about more than twenty types of histone PTMs exists, little is known about histone PTM combinations (PTMCs). Due to the combinatorial explosion it is intrinsically impossible to consider all potential PTMCs in a database search. Consequentially, high-scoring false positives with unconsidered but correct alternative isobaric PTMCs can occur. Current quality controls can neither estimate the amount of unconsidered alternatives nor flag potential false positives. Here, we propose a conceptual workflow that provides such options. In this workflow, an in silico modelling of all candidate isoforms with known-to-exist PTMs is made. The most frequently occurring PTM sets of these candidate isoforms are determined and used in several database searches. This suppresses the combinatorial explosion while considering as many candidate isoforms as possible. Finally, annotations can be classified as unique or ambiguous, the latter implying false positives. This workflow was evaluated on an LCMS dataset containing 44 histone extracts. We were able to consider 60% of all candidate isoforms. Importantly, 40% of all annotations were classified as ambiguous. This highlights the need for a more thorough evaluation of modified peptide annotations

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. Employing complementary techniques, different cleavage sites could be identified, namely A21, R26 and residue 31. The enzyme responsible for this cleavage is found to be a serine protease. The formation of cleaved H3 follows a considerably variable pattern, depending on the timeframe, culture conditions and culture media applied. Contrary to earlier findings on H3 clipping, our results disconnect the link between declining Oct4 expression and H3 cleavage