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.

L2hgdh Deficiency Accumulates l-2-Hydroxyglutarate with Progressive Leukoencephalopathy and Neurodegeneration

“…We therefore performed multiple reaction monitoring (MRM) based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis with isotopically labeled standards to quantify relative histone levels (23,24). Our data revealed that H3K9me2 and H3K9me3 levels were significantly increased while H3K9me0 levels were reduced in the testis of L2hgdh KO mice (Fig.…”

“…Histone methylation was quantified by MRM-based LC-MS/MS (LC-MRM-MS), as described previously (23,24). In short, the core histones were acid extracted from tissue with 0.4 M H 2 SO 4 and precipitated with trichloroacetic acid (TCA), followed by 2 washes with ice-cold acetone.…”

Analysis of contribution of protein phosphorylation in the development of the diseases

“…Но благодаря аффинному обогащению модифицированных белков/пептидов и масс-спектрометрии исследования этих ПТМ получили свое развитие. Опубликованы множество работ, в которых количественно сравниваются профили ацетилирования [87][88][89][90][91], метилирования [87][88][89][90]92], убиквитинирования [88,[93][94][95][96] и гликозилирования [97][98][99] белков нормальных и раковых клеток. Достоверно установлено, что ацетилирование регулирует доступ транскрипционных белков к промоторам на ДНК за счёт структурного изменения хроматина.…”

In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana

“…), a 550 °C interface heater temperature, collision Gas set to high, and nebulizer gas (gas 1) and heater gas M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 9 (gas 2) were set at 65 p.s.i.. MRM was employed, with two transitions for exogenous histone and isotope labeled peptides. The parameter (DP, CE,Q1,Q2) of MRM method followed a publish work with some modifications to obtain maximum MRM intensity (Table S1) [14].…”

“…Mass spectrometry (MS) has proved to be a robust and powerful tool to analyze histones as it can simultaneously be used to identify and quantify histone variants and PTMs [11][12][13][14]. Though, in solution derivatization and digestion has been widely applied in proteomic research, which is suitable for large scale investigation with high reproducibility, many proteomics researches are based on the gel system [15][16][17].…”