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

Tyrosine-Directed Conjugation of Large Glycans to Proteins via Copper-Free Click Chemistry

We have demonstrated that the insertion of alkyne-containing bifunctional linkers into the tyrosine residues of the carrier protein, followed by the copper mediated azide–alkyne [3 + 2] cycloaddition of carbohydrates, is a robust approach for the preparation of glycoconjugates with defined glycans, carrier, and connectivity. Conjugation of Group B Streptococcus (GBS) capsular polysaccharides to streptococcal pilus protein could extend the vaccine coverage to a variety of strains. Application of our protocol to these large charged polysaccharides occurred at low yields. Herein we developed a tyrosine-directed conjugation approach based on the copper-free click chemistry of sugars modified with cyclooctynes, which enables efficient condensation of synthetic carbohydrates. Most importantly, this strategy was demonstrated to be more effective than the corresponding copper catalyzed reaction for the insertion of GBS onto the tyrosine residues of GBS pilus proteins, previously selected as vaccine antigens through the so-called <i>reverse vaccinology</i>. Integrity of protein epitopes in the modified proteins was ascertained by competitive ELISA, and conjugation of polysaccharide to protein was confirmed by SDS page electrophoresis and immunoblot assays. The amount of conjugated polysaccharide was estimated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The described technology is particularly suitable for proteins used with the dual role of vaccine antigen and carrier for the carbohydrate haptens

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