Abstract
A two dimensional-liquid chromatography (2D-LC) based approach was developed for the identification and quantification of histone post translational modifications in conjunction with mass spectrometry analysis. Using a bottom-up strategy, offline 2D-LC was developed using reverse phase chromatography. A porous graphitic carbon stationary phase in the first dimension and a C18 stationary phase in the second dimension interfaced with mass spectrometry was used to analyse global levels of histone post translational modifications in human primary monocyte-derived macrophages. The results demonstrated that 84 different histone peptide proteoforms, with modifications at 18 different sites including combinatorial marks were identified, representing an increase in the identification of histone peptides by 65% and 51% compared to two different 1D-LC approaches on the same mass spectrometer.The use of the porous graphitic stationary phase in the first dimension resulted in efficient separation of histone peptides across the gradient, with good resolution and is orthogonal to the online C18 reverse phase chromatography. Overall, more histone peptides were identified using the 2D-LC approach compared to conventional 1D-LC approaches. In addition, a bioinformatic pipeline was developed in-house to enable the high throughput efficient and accurate quantification of fractionated histone peptides. The automation of a section of the downstream analysis pipeline increased the throughput of the 2D-LC–MS/MS approach for the quantification of histone post translational modifications.
Highlights
Histone post translational modifications (PTMs) play key roles in regulating eukaryotic gene expression
Histone proteins were purified from 14 day old human primary macrophages by acid extraction. 10 g of histone protein was subjected to chemical dervitisation by propionic anhydride prior to and post tryptic digestion
One minute fractions were collected, dried and analysed using nano-flow reverse phase chromatography on a C18 stationary phase interfaced to mass spectrometry
Summary
Histone post translational modifications (PTMs) play key roles in regulating eukaryotic gene expression. Histone N-terminal tails are key targets for PTMs since they protrude from the nucleosome and can make contact with adjacent nucleosomes, providing a mechanism for regulating regional protein-DNA and protein–protein interactions. The PTM of histones provide binding sites for a number of effector molecules that can establish and orchestrate downstream events [3,4,5]. These histone marks dictate chromatin structure but they control access to the underlying DNA and are involved in all DNA based processes including gene expression [3]
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