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Mol Cell Proteomics. 2014 Sep;13(9):2450-66. doi: 10.1074/mcp.O113.036459. Epub 2014 Jul 7.

Stable-isotope-labeled histone peptide library for histone post-translational modification and variant quantification by mass spectrometry.

Author information

1
From the ‡Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
2
From the ‡Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; §Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544;
3
¶Epigenetics Program, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
4
‖Department of Protein Chemistry, Genentech Inc., South San Francisco, California 94080;
5
**Cell Signaling Technology Inc., Danvers, Massachusetts 01923.
6
From the ‡Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; bgarci@mail.med.upenn.edu.

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.

PMID:
25000943
PMCID:
PMC4159661
DOI:
10.1074/mcp.O113.036459
[Indexed for MEDLINE]
Free PMC Article
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