Specific and Efficient N-propionylation of histones with Propionic acid N-hydroxysuccinimide Ester for Histone Marks Characterization by LC-MS

Histones participate in transcriptional regulation via a variety of dynamic posttranslational modifications (PTMs) on them. Mass spectrometry (MS) has become a powerful tool to investigate histone PTMs. With the Bottom-up mass spectrometry approach, chemical derivatization of histones with propionic anhydride or deuterated acetic anhydride followed by trypsin digestion was widely used to block the hydrophilic lysine residues and generates compatible peptides for LC-MS analysis. However, some serious side reactions (such as acylation on tyrosine or serine) caused by acid anhydrides will lead to a number of analytical problems such as reducing the accuracy and impairing reproducibility and sensitivity of analysis. Thereby we report a novel derivatization method that utilizes N-HydroxySuccinimide ester to specifically and efficiently derivatize both free and monomethylated amine groups in histones. A competitive inhibiting strategy was implemented in our method to effectively avoid the side reactions. We demonstrated that our method can achieve excellent specificity and efficiency for histones derivatization in a reproducible manner. To test in vivo samples, we applied the derivatization method to quantitatively profile the histone PTMs in the KMS11 cell line with selective knock out of the translocated NSD2 (a histone methyltransferase that catalyzes the histone H3 lysine 36 methylation) and its parental cells. Comparative quantification revealed a significant crosstalk between H3 protein K27 methylation and adjacent K36 methylation

Specific and Efficient N‑Propionylation of Histones with Propionic Acid <i>N</i>‑Hydroxysuccinimide Ester for Histone Marks Characterization by LC-MS

Histones participate in epigenetic regulation via a variety of dynamic posttranslational modifications (PTMs) on them. Mass spectrometry (MS) has become a powerful tool to investigate histone PTMs. With the bottom-up mass spectrometry approach, chemical derivatization of histones with propionic anhydride or deuterated acetic anhydride followed by trypsin digestion was widely used to block the hydrophilic lysine residues and generate compatible peptides for LC-MS analysis. However, certain severe side reactions (such as acylation on tyrosine or serine) caused by acid anhydrides will lead to a number of analytical issues such as reducing results accuracy and impairing the reproducibility and sensitivity of MS analysis. As an alternative approach, we report a novel derivatization method that utilizes <i>N</i>-hydroxysuccinimide ester to specifically and efficiently derivatize both free and monomethylated amine groups in histones. A competitive inhibiting strategy was implemented in our method to effectively prevent the side reactions. We demonstrated that our method can achieve excellent specificity and efficiency for histones derivatization in a reproducible manner. Using this derivatization method, we succeeded to quantitatively profile the histone PTMs in KMS11 cell line with selective knock out of translocated NSD2 allele (TKO) and the original parental KMS11 cell lines (PAR) (NSD2, a histone methyltransferase that catalyzes the histone H3 K36 methylation), which revealed a significant crosstalk between H3 protein K27 methylation and adjacent K36 methylation

The PHD domain targets NSD2 to oncogenic gene loci and drives tumorigenesis in multiple myeloma cells

NSD2, a histone lysine methyltransferase, is overexpressed as a result of the t(4;14) translocation that is associated with 15-20% of multiple myeloma patients. Earlier studies have indicated that NSD2 may be involved in myelomagenesis and suggested that it may be a target for myeloma therapy. Here we show that depletion of NSD2 has only minor influence on the proliferation of t(4;14)+ myeloma cells. However, we found that NSD2 is required for clonogenic growth, adherence and proliferation on bone marrow stroma, and tumorigenesis of t(4;14)+ but not t(4;14)- myeloma cells, in a methyltransferase activity dependent manner. Furthermore, we found that PHD domains are important for NSD2 cellular activity and biological functions in myeloma by recruiting it to oncogenic gene loci and driving downstream transcription activation events. These results strengthened the disease link of NSD2 and provided a basis that targeting the methyltransferase activity of NSD2 may be a therapeutic strategy in multiple myeloma patients with t(4;14) translocation. Our data also revealed multiple domains in the protein for possible chemical modulation

Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS

Deciphering the combinatorial histone codes has been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the post-translational modifications (PTMs) coexisting on histones. To this end, weak cation exchange hydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography–mass spectrometry approaches for online separation of variously modified histone peptides. Here we provide a novel strategy that combines the selective histone peptide derivatization using <i>N</i>-hydroxysuccinimide propionate ester with reversed-phase liquid chromatography (RPLC) for the robust, sensitive, and reliable characterization of combinatorial histone PTMs. Derivatization amplifies the subtle physical differences between similarly modified histone peptides, thereby allowing baseline separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatization due to the increased peptide hydrophobicity and concentrated charge-state envelope during electrospray ionization. Furthermore, we systematically optimized the dual electron transfer and higher energy collision dissociation and achieved near-complete peptide sequence coverage in MS/MS spectra, allowing highly precise and reliable PTM identification. Using this method, we identified 311 and 293 combinations of histone H3 PTMs from the lymphoma cells Karpas-422 with/without drug treatment, confirming the advantages of our method in serving as a platform for profiling combinatorial histone PTMs

Absolute Quantification of Histone PTM Marks by MRM-Based LC-MS/MS

The N-terminal tails of core histones harbor the sites of numerous post-translational modifications (PTMs) with important roles in the regulation of chromatin structure and function. Profiling histone PTM marks provides data that help understand the epigenetics events in cells and their connections with cancer and other diseases. Our previous study demonstrated that specific derivatization of histone peptides by NHS propionate significantly improved their chromatographic performance on reversed phase columns for LC/MS analysis. As a step forward, we recently developed a multiple reaction monitoring (MRM) based LC-MS/MS method to analyze 42 targeted histone peptides. By using stable isotopic labeled peptides as internal standards that are spiked into the reconstituted solutions, this method allows to measure absolute concentration of the tryptic peptides of H3 histone proteins extracted from cancer cell lines. The method was thoroughly validated for the accuracy and reproducibility through analyzing recombinant histone proteins and cellular samples. The linear dynamic range of the MRM assays was achieved in 3 orders of magnitude from 1 nM to 1 μM for all targeted peptides. Excellent intrabatch and interbatch reproducibility (<15% CV) was obtained. This method has been used to study translocated NSD2 (a histone lysine methyltransferase that catalyzes the histone lysine 36 methylation) function with its overexpression in KMS11 multiple myeloma cells. From the results we have successfully quantitated both individual and combinatorial histone marks in parental and NSD2 selective knockout KMS11 cells