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. 2010 May;9(5):824-37.
doi: 10.1074/mcp.M900569-MCP200. Epub 2010 Feb 4.

High resolution electron transfer dissociation studies of unfractionated intact histones from murine embryonic stem cells using on-line capillary LC separation: determination of abundant histone isoforms and post-translational modifications

Shannon M Eliuk  1 , David Maltby, Barbara Panning, Alma L Burlingame
Affiliations

High resolution electron transfer dissociation studies of unfractionated intact histones from murine embryonic stem cells using on-line capillary LC separation: determination of abundant histone isoforms and post-translational modifications

Shannon M Eliuk et al. Mol Cell Proteomics. 2010 May.

Abstract

Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed. From a single LC-electron transfer dissociation run, this strategy permits the identification of the most abundant intact proteins, determination of the isoforms present, and the localization of post-translational modifications.

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Figures

Fig. 1.
Fig. 1.
Protein identification work flow for sample preparation and data analysis for determination of protein isoform and localization of PTMs.
Fig. 2.
Fig. 2.
On-line RP UPLC separation of intact, unfractionated histones. a, total ion chromatogram; b, base peak ion chromatogram; and c, extracted ion chromatograms for the most intense ion in each chromatographic peak. The deconvoluted, monoisotopic masses are indicated for the most abundant species in each peak.
Fig. 3.
Fig. 3.
Determination of histone H2B isoform by on-line LC Orbitrap ETD-MS/MS analysis. a, sequence alignment for mouse histone H2B types 1-M, 1-F, and 3-B. Sites of sequence variation are indicated in red. b, ETD fragment ion spectrum with only the most abundant ions labeled. The spectrum is an average spectrum composite of three MS/MS spectra. c, fragment ion assignments for histone H2B type 1-F. Although three isobaric H2B variants exist, ETD fragmentation enables the detection and confident identification of histone H2B type 1-F.
Fig. 4.
Fig. 4.
On-line Orbitrap MS of histone H4 molecular ions. a, broadband FT mass spectrum; b, 18+ base peak with ETD isolation window indicated; and c, the deconvoluted histone H4 spectrum. The high resolution mass spectrum acquired in the Orbitrap enables charge state and monoisotopic mass determination necessary for accurate intact protein identification.
Fig. 5.
Fig. 5.
On-line LC-Orbitrap ETD-MS/MS analysis of intact histone H4 for localization of PTMs. a, ETD fragment ion spectrum; only the most abundant ions are labeled. The spectrum is an average spectrum composite of four MS/MS spectra. b, labeled ETD fragment ions for the mass range of m/z 820–940. c, fragment ion assignments. ETD fragmentation enables the determination of N-terminal loss of methionine and localization of monoacetylation to the N terminus and dimethylation at Lys-20.
Fig. 6.
Fig. 6.
Extracted ion chromatograms showing typical separation of differentially modified histone H4 N-terminal tails according to level of acetylation. An increased level of acetylation increased the peptide retention time of the peptides; however, the level of methylation did not affect the retention time. Although signal to noise is high for the extracted ion chromatograms of peptides with five acetylations, the inset shows the sufficient ion intensity in the MS spectrum.
Fig. 7.
Fig. 7.
LC-ETD MS/MS analysis of modified histone H4 N-terminal peptides following unfractionated histone Asp-N digestion. a, upper panel, high resolution ETD fragment ion spectrum of the histone H4 N-terminal tail with the addition of three acetylations and two methylations; lower panel, m/z range of 600–650. b, detected fragment ion assignments with variable modifications indicated.
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