Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry

Abstract

To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono-(me1), di-(me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the uncorrected data. The peptide library and detection efficiencies presented here may serve as a resource to facilitate studies in the epigenetics and proteomics fields. In eukaryotes, histones package and order DNA into nucleosomes. Histones are critical for the structural organization and transcriptional activities of chromatin. These proteins are highly conserved in eukaryotes, but they have very dynamic functions and are subject to many different regulatory mechanisms (1). One of the major mechanisms is enacted via the use of different histone variants and post-translational modifications (PTMs). 1Over the past two decades, accumulating evidence has suggested that levels and PTM compositions of many histone variants play important roles in cellular processes and human diseases. There are increasing needs for the precise quantification of histone variants and PTMs in biomedical studies. Mass spectrometry (MS) has proved to be a robust and powerful tool for analyzing histones, as it can be used to simultaneously identify and quantify histone variants and ...

EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra*

“…We observed that the proportion calculated by MS1 of H3K9ac was usually lower than the input concentration. We therefore corrected for this detection efficiency as previously described (17). All the corrected proportions were very close to the input (as shown in Fig.…”

“…Synthetic Heavy-labeled Histone Peptides-Synthetic peptides were purchased via Cell Signaling Technology Protein-Aqua (MA) as described (17). Briefly, a library of 93 synthetic histone peptides was assembled, including the most abundant modified and unmodified forms.…”

“…However, determination of peptide RT might be difficult because of their low abundance though acid extraction was performed to purify histones. This problem can be solved by using isotopically labeled synthetic histone peptides (17), or data independent approaches (18). When using relative retention time information to assign peak identities, reproducible nanoLC is crucial, especially because some isobaric peptides co-elute.…”

Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells

“…Subsequently, we determined the relative abundance at the MS1 level based on the ratios we obtained, either manually or by a Matlab-based program, EpiProfile (69). Data were corrected for differences in detection efficiencies based on the correction factors generated by a synthetic peptide library as described previously (70). For phosphorylated peptides that were not included in the synthetic peptide library, an average correction factor generated from all the peptides with the same histone backbone was used.…”

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

“…Moreover, it allows better discrimination of histone variants, which cannot always be achieved with bottom-up since some peptides have the same sequence in different histone variants. The second issue, related to the ionization efficiency, can be solved using a library of synthetic peptides 31 . This approach ensures a more accurate estimation of the relative abundance of histone PTMs.…”