title: Absolute quantification of histone PTM marks by MRM-based LC-MS/MS.
abstract: 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.
authors.affiliation: China Novartis Institutes for BioMedical Research Co. Ltd. , Building 8, Lane 898 Halei Road, Shanghai 201203, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Gao
authors.forename: Rijing
authors.initials: R
authors.lastname: Liao
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Yu
authors.forename: Huili
authors.initials: H
authors.lastname: Zhai
authors.forename: Yingqi
authors.initials: Y
authors.lastname: Wang
authors.forename: Ragna
authors.initials: R
authors.lastname: Sack
authors.forename: Antoine H F M
authors.initials: AH
authors.lastname: Peters
authors.forename: Jiajia
authors.initials: J
authors.lastname: Chen
authors.forename: Haiping
authors.initials: H
authors.lastname: Wu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Huang
authors.forename: Min
authors.initials: M
authors.lastname: Hu
authors.forename: Wei
authors.initials: W
authors.lastname: Qi
authors.forename: Chris
authors.initials: C
authors.lastname: Lu
authors.forename: Peter
authors.initials: P
authors.lastname: Atadja
authors.forename: Counde
authors.initials: C
authors.lastname: Oyang
authors.forename: En
authors.initials: E
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Yi
authors.forename: Shaolian
authors.initials: S
authors.lastname: Zhou
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006657:Histones / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006657:Histones