Validation of an LC‐MS based approach for profiling histones in chronic lymphocytic leukemia

Abstract

The in vitro evaluation of histones and their post-translational modifications has drawn substantial interest in the development of epigenetic therapies. The differential expression of histone isoforms may serve as a potential marker in the classification of diseases affected by chromatin abnormalities. In this study, protein profiling by liquid chromatography and mass spectrometry was used to explore differences in histone composition in primary CLL cells. Extensive method validations were performed to determine the experimental variances that would impact histone relative abundance. The resulting data demonstrated that the proposed methodology was suitable for the analysis of histone profiles. In 4 normal individuals and 40 CLL patients, a significant decrease in the relative abundance of histone H2A variants (H2AFL and H2AFA/M*) was observed in primary CLL cells as compared to normal B cells. Protein identities were determined using high mass accuracy mass spectrometry and shotgun proteomics.

Ultra-High Performance Liquid Chromatography−Mass Spectrometry for the Fast Profiling of Histone Post-Translational Modifications

“…These values are in excellent agreement with the ones of 9 ppm (for H2A) 13 and 5 ppm (for H4) 19 previously obtained on Orbitrap and FT-ICR instruments, respectively. Such mass accuracy corresponds to a ∼0.1 Da error in mass, which is ∼10 fold better than the average ∼1 Da mass error previously reported for Q-TOF instruments.…”

“…Such mass accuracy corresponds to a ∼0.1 Da error in mass, which is ∼10 fold better than the average ∼1 Da mass error previously reported for Q-TOF instruments. 13,27 One of the strengths of such protein profiling approach is the potential comprehensive characterization of abundant PTMs and variants. 29 Since core histones exhibit distinct charge state distributions, the selection of two different precursor ions with a m/z 70 isolation window was required to efficiently and automatically fragment all core histone species in a single experiment.…”

Histone Variants and Their Post-Translational Modifications in Primary Human Fat Cells

“…For example, a significant decrease in the relative abundance of histone H2A variants H2AFL and H2AFA/M was observed in primary chronic lymphocytic leukemia cells as compared to normal B cells [61]. Mammalian histones show tissue-specific differences in their expression and PTMs [62], as well as pronounced differences between cell cultures and primary tissues [29].…”

Breaking the histone code with quantitative mass spectrometry

“…Offline reverse-phase HPLC has been widely used to separate out bulk histone variants, particularly in the cases of H2As, H3s and H1s, which often can resolve into separate peaks. Commonly, these separations have been coupled on-line to MS analysis, making intact protein profiling of histones a rapid and facile experiment [96]. Thorough characterization of histone variants by top-down MS has been performed on all histones to date.…”