Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence

Yu, Chang; Hoover, Michael E.; Dang, Xibei; Shomo, Alan A.; Guan, Xiaoyan; Marshall, Alan G.; Freitas, Michael A.; Young, Nicolas L.

doi:10.1074/mcp.m114.046441

Cited by 30 publications

(25 citation statements)

References 75 publications

(91 reference statements)

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“…The results of several recent studies involving top-down analyses of histones highlight the complexity of the histone proteome as well as important biological implications that are not easily captured unless the proteoforms are analyzed intact (17)(18)(19)(20). Although these results are very promising, this approach is not routinely utilized because of the many challenges with which it is associated.…”

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confidence: 91%

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Anderson

Karch

Ugrin

et al. 2016

Molecular & Cellular Proteomics

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Chromatin is the structural framework that packages DNA into chromosomes within the nucleus of a cell (2). Histones comprise the principal protein component of chromatin and are involved in the regulation of gene expression (3,4). This epigenetic regulation is achieved through complex patterns of post-translational modifications (PTMs), 1 the incorporation of histone variants, and through controlled histone proteolysis (5-10). Comprehensive characterization of histones by mass spectrometry (MS) has proven technically difficult for a number of reasons. Traditional methods (bottom-up MS) of sequence determination and PTM site localization are not practical. Histone N-terminal regions are rich in lysine and arginine residues, and thus proteolysis using trypsin generates peptides that are too small or that are poorly retained on reversephase HPLC C18 resins for subsequent MS detection (11). With the advent of electron transfer dissociation (ETD) and more efficient electron capture dissociation fragmentation methods, which are better suited for larger, more highly charged peptides (12, 13), several studies utilizing other endoproteases to generate longer peptides have emerged (14 -16). Although these methodologies do well to preserve the combinatorial PTM profiles of histone tails, in some cases it is still impossible to identify the proteoforms from which these peptides originate. This is why analyzing histones intact, as they exist in the cells from which they are derived, is the best method for identifying unique histone proteoforms.The results of several recent studies involving top-down analyses of histones highlight the complexity of the histone From the ‡Department

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confidence: 91%

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Anderson

Karch

Ugrin

et al. 2016

Molecular & Cellular Proteomics

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“…Mazur et al determined differences in the abundance of human apolipoproteins using the peak areas of precursor ( m / z ) extracted ion chromatograms 12 . Phosphorylated proteoform abundance has been measured using peak intensities of the top 5 isotopomers; 13 or the most intense isotopic peak 14 normalized by the total intensity of all the observed proteoforms. Wu et al compared the abundance of human saliva proteins from the parotid gland and submandibular/ sublingual gland using the sum of extracted ion chromatogram peak areas of a proteoform’s charge states normalized by the total ion intensity 15 .…”

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confidence: 99%

Evaluation of Spectral Counting for Relative Quantitation of Proteoforms in Top-Down Proteomics

et al. 2016

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Spectral counting is a straightforward label-free quantitation strategy used in bottom-up proteomics workflows. The application of spectral counting in label-free top-down proteomics workflows can be similarly straightforward but has not been applied as widely as quantitation by chromatographic peak areas or peak intensities. In this study, we evaluate spectral counting for quantitative comparisons in label-free top down proteomics workflows by comparison with chromatographic peak areas and intensities. We tested these quantitation approaches by spiking standard proteins into a complex protein background and comparing relative quantitation by spectral counts with normalized chromatographic peak areas and peak intensities from deconvoluted extracted ion chromatograms of the spiked proteins. Ratio estimates and statistical significance of differential abundance from each quantitation technique are evaluated against the expected ratios and each other. In this experiment, spectral counting was able to detect differential abundance of spiked proteins for expected ratios ≥2, with comparable or higher sensitivity than normalized areas and intensities. We also found that while ratio estimates using peak areas and intensities are usually more accurate, the spectral-counting-based estimates are not substantially worse. Following the evaluation and comparison of these label-free top down quantitation strategies using spiked proteins, spectral counting, along with normalized chromatographic peak areas and intensities, were used to analyze the complex protein cargo of exosomes shed by myeloid-derived suppressor cells collected under high and low conditions of inflammation, revealing statistically significant differences in abundance for several proteoforms, including the active pro-inflammatory proteins S100A8 and S100A9.

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“…Both MCF7 (expressing an allelic variant A142T) and MDA231, have a greater number of histone H1.2 phosphorylations when compared to MCF10A cell line 22 . Curiously, phosphorylation of histone H1.2 at S173 increases during the M phase relative to the S phase, suggesting that this event is cell cycle-dependent and may serve as a marker for proliferation of cancer cells during BC invasion 23 , 24 . Also, histone H1.2 is a novel component of the nucleolar organizer regions during mitosis 25 and H1.2 depletion was observed in a human BC cell line caused cell cycle G1-phase arrest 26 .…”

Section: Resultsmentioning

confidence: 99%

Blood-derived non-extracellular vesicle proteins as potential biomarkers for the diagnosis of early ER+ breast cancer and detection of lymph node involvement

Tucker¹,

Pedro²

2018

F1000Res

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Extracellular vesicles (EV’s) are membrane surrounded structures released by different cell types and are emerging as potential therapeutic and diagnostic targets in cancer. In the present study, plasma samples derived from 7 patients with metastatic and non-metastatic ER+ (estrogen receptor positive) breast cancer (BC) were collected and their respective (EVs) isolated and the protein content analyzed by mass spectrometry and FunRich analysis. Two putative plasma biomarkers (absent in healthy controls samples) were identified which could be used to detect early ER+ breast cancer and for those with lymph node (LN) involvement However, given the current limitations of the EV isolation method used, it is possible that these biomarkers did not originate from EVs and may represent blood-derived extracellular proteins. Further work in a larger patient cohort is warranted to confirm these findings and examine the diagnostic potential of these biomarkers.

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Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence

Cited by 30 publications

References 75 publications

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Evaluation of Spectral Counting for Relative Quantitation of Proteoforms in Top-Down Proteomics

Blood-derived non-extracellular vesicle proteins as potential biomarkers for the diagnosis of early ER+ breast cancer and detection of lymph node involvement

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