Quantification of Histone Modifications by Parallel-Reaction Monitoring: A Method Validation

James L. Sowers, Barsam Mirfattah, Pei Xu, Hui Tang, In Young Park, Cheryl Walker, Ping Wu, Fernanda Laezza, Lawrence Sowers, Kangling Zhang

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Abnormal epigenetic reprogramming is one of the major causes leading to irregular gene expression and regulatory pathway perturbations, in the cells, resulting in unhealthy cell development or diseases. Accurate measurements of these changes of epigenetic modifications, especially the complex histone modifications, are very important, and the methods for these measurements are not trivial. By following our previous introduction of PRM to targeting histone modifications (Tang, H.; Fang, H.; Yin, E.; Brasier, A. R.; Sowers, L. C.; Zhang, K. Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer. Anal. Chem. 2014, 86 (11), 5526-34), herein we validated this method by varying the protein/trypsin ratios via serial dilutions. Our data demonstrated that PRM with SILAC histones as the internal standards allowed reproducible measurements of histone H3/H4 acetylation and methylation in the samples whose histone contents differ at least one-order of magnitude. The method was further validated by histones isolated from histone H3 K36 trimethyltransferase SETD2 knockout mouse embryonic fibroblasts (MEF) cells. Furthermore, histone acetylation and methylation in human neural stem cells (hNSC) treated with ascorbic acid phosphate (AAP) were measured by this method, revealing that H3 K36 trimethylation was significantly down-regulated by 6 days of treatment with vitamin C.

Original languageEnglish (US)
Pages (from-to)10006-10014
Number of pages9
JournalAnalytical Chemistry
Volume87
Issue number19
DOIs
StatePublished - Oct 6 2015

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Histones
Monitoring
Acetylation
Methylation
Ascorbic Acid
Mass spectrometers
Fibroblasts
Stem cells
Gene expression
Trypsin
Dilution
Phosphates
Cells

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Quantification of Histone Modifications by Parallel-Reaction Monitoring : A Method Validation. / Sowers, James L.; Mirfattah, Barsam; Xu, Pei; Tang, Hui; Park, In Young; Walker, Cheryl; Wu, Ping; Laezza, Fernanda; Sowers, Lawrence; Zhang, Kangling.

In: Analytical Chemistry, Vol. 87, No. 19, 06.10.2015, p. 10006-10014.

Research output: Contribution to journalArticle

Sowers, James L. ; Mirfattah, Barsam ; Xu, Pei ; Tang, Hui ; Park, In Young ; Walker, Cheryl ; Wu, Ping ; Laezza, Fernanda ; Sowers, Lawrence ; Zhang, Kangling. / Quantification of Histone Modifications by Parallel-Reaction Monitoring : A Method Validation. In: Analytical Chemistry. 2015 ; Vol. 87, No. 19. pp. 10006-10014.
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