Extracting histones for the specific purpose of label‐free MS

Abstract

Extracting histones from cells is the first step in studies that aim to characterize histones and their post‐translational modifications (hPTMs) with MS. In the last decade, label‐free quantification is more frequently being used for MS‐based histone characterization. However, many histone extraction protocols were not specifically designed for label‐free MS. While label‐free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label‐free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS‐PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label‐free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885.

Untargeted histone profiling during naive conversion uncovers conserved modification markers between mouse and human

“…After assessment of the suitability of different extraction protocols 45 , nuclei were isolated from frozen cell pellets by resuspension in hypotonic lysis buffer (HLB). First, 2.10 6 cells were resuspended in 400 µl of HLB (10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl2) supplemented with 1 mM DTT, Halt protease and phosphatase inhibitor cocktail EDTA-free (788441, Thermo Fisher, 1 mL of cocktail for 100 mL of buffer) and phosphatase inhibitor cocktails II and III (P5726 and P0044, Sigma-Aldrich, 1 mL of cocktail for 100 mL of buffer) and incubated for 30 minutes on a rotator at 4 °C.…”

Simultaneous Improvement in the Precision, Accuracy, and Robustness of Label-free Proteome Quantification by Optimizing Data Manipulation Chains*

“…The proteomics datasets of the third through fifth studies (59 -61) were acquired by the DDA (peak intensity) technique and quantified by MaxQuant. Meanwhile, the datasets from the sixth through eighth studies (54,62,63) were acquired by DDA (peak intensity) technique and quantified by Progenesis. These six studies therefore resulted in six benchmark datasets.…”

Variations of Histone Modification Patterns: Contributions of Inter-plant Variability and Technical Factors

“…Increased protein complexity given especially by higher proportions of co-extracting ribosomal proteins subsequently affected detection of histone peptides during the MS/MS experiment. Similarly, histone extraction protocols designed to minimize handling of human cell culture samples were recently found to increase levels of co-extracted ribosomal proteins, but not to affect RA-based histone peptide quantification ( Govaert et al, 2016 ). Accordingly, the RAs of selected histone peptide forms obtained from analyses of A. thaliana extracts prepared using our automated and manual homogenization procedures were similar, despite the difference in the purity of histone extracts.…”