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. 2016 Dec;16(23):2937-2944.
doi: 10.1002/pmic.201600341.

Extracting Histones for the Specific Purpose of Label-Free MS

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Free PMC article

Extracting Histones for the Specific Purpose of Label-Free MS

Elisabeth Govaert et al. Proteomics. .
Free PMC article

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.

Keywords: Comparative study; Epigenetics; Extraction protocol; Histone; Label-free MS quantification; Technology.

Figures

Figure 1
Figure 1
Overview of the different histone extraction protocols, highlighting the differences between Protocols A, B, and C.
Figure 2
Figure 2
Extraction efficiency evaluation of the different protocols using SDS‐PAGE based purity ratios. (A) Purity of histone extracts prepared using Protocols A, B, and C on fresh and snap‐frozen cells for Jurkat cells and hESC, as compared to bovine histones, a commercial extract obtained after extensive fractionation. Independent technical replicates n = 3, mean ± SD. Replicates were prepared in different tubes at different time points by the same person. (B) Four protocols were selected for subsequent MS analysis and performed on Jurkat cells.
Figure 3
Figure 3
Feature‐level based analysis of the different protocols. (A) Based on MS precursor intensities (gray cloud), a PCA was performed resulting in the clustering of Protocol C‐fresh (Cluster 1: SDC) versus Protocols A‐fresh, A‐frozen and B‐frozen (Cluster 2: No SDC) along PC 1. Color code: A‐fresh (blue), C‐fresh (purple), A‐frozen (orange), B‐frozen (green). (B) Distribution of corrected CV values obtained for independent technical replicates (n = 6–8) shown as violin plots. The median CV is depicted by a red dot.
Figure 4
Figure 4
Label‐free quantification of histone variants and hPTMs. (A) Table depicting significantly different extracted histone variants (q‐value < 0.01) between the four protocols. Note that H2AX_HUMAN was only quantified by one unique peptide. (B) Radar charts representing the average RA of targeted hPTMs on two H3 peptides and one H4 peptide. Each targeted hPTM is located on one angle of the radar chart and each protocol is represented by a different color A‐fresh (blue), C‐fresh (purple), A‐frozen (orange), B‐frozen (green) whereby a RA of 0 is located in the center, ascending outwards. RAs and their SDs can be consulted in Supporting Information Table 2. K, lysine; R, arginine; S, serine; un, unmodified; me1, monomethylation; me2, dimethylation; me3, trimethylation; ac, acetylation; ph, phosphorylation

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