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High-Throughput Quantitative Top-Down Proteomics: Histone H4

  • Matthew V. Holt
  • Tao Wang
  • Nicolas L. YoungEmail author
Focus: 34th Asilomar Conference, Quantitative Analysis of PTMs: Research Article

Abstract

Proteins physiologically exist as “proteoforms” that arise from one gene and acquire additional function by post-translational modifications (PTM). When multiple PTMs coexist on single protein molecules, top-down proteomics becomes the only feasible method of characterization; however, most top-down methods have limited quantitative capacity and insufficient throughput to truly address proteoform biology. Here we demonstrate that top-down proteomics can be quantitative, reproducible, sensitive, and high throughput. The proteoforms of histone H4 are well studied both as a challenging proteoform identification problem and due to their essential role in the regulation of all eukaryotic DNA-templated processes. Much of histone H4’s function is obfuscated from prevailing methods due to combinatorial mechanisms. Starting from cells or tissues, after an optimized protein purification process, the H4 proteoforms are physically separated by on-line C3 chromatography, narrowly isolated in MS1 and sequenced with ETD fragmentation. We achieve more than 30 replicates from a single 35-mm tissue culture dish by loading 55 ng of H4 on column. Parallelization and automation yield a sustained throughput of 12 replicates per day. We achieve reproducible quantitation (average biological Pearson correlations of 0.89) of hundreds of proteoforms (about 200–300) over almost six orders of magnitude and an estimated LLoQ of 0.001% abundance. We demonstrate the capacity of the method to precisely measure well-established changes with sodium butyrate treatment of SUM159 cells. We show that the data produced by a quantitative top-down method can be amenable to parametric statistical comparisons and is capable of delineating relevant biological changes at the full proteoform level.

Keywords

Histone post-translational modifications Histone proteoforms Dynamics of histone modifications Epigenetic inhibitor Top-down proteomics 

Notes

Acknowledgements

We would like to thanks Dr. A. Assié for critical reviews on the manuscript.

Raw data and full proteoform lists:

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [46] partner repository with the dataset identifier PXD013766

Supplementary material

13361_2019_2350_MOESM1_ESM.pptx (118 kb)
Supplementary Figure 1. Normalized extracted ion chromatograms of 755.83 m/z (Nα-acK20me2M84ox). Black: technical replicates of biological replicate 1. Red: technical replicates of biological replicate 2. Blue: technical replicates of biological replicate 3. Orange: technical replicates of biological replicate 4. (PPTX 117 kb)
13361_2019_2350_MOESM2_ESM.pptx (61 kb)
Supplementary Figure 2. Offline HPLC UV standard curve for the quantitation of histone H4 present in each sample based on recombinant Xenopus laevis H4. (PPTX 60 kb)
13361_2019_2350_MOESM3_ESM.pptx (115 kb)
Supplementary Figure 3. A) A scatter plot of CVs vs. log10 of % abundance. Black: biological replicates, Red: average of technical replicates. B) Boxplots of the top 100 proteoform CVs. Technical CVs are significantly lower than biological (p<0.01) (PPTX 114 kb)
13361_2019_2350_MOESM4_ESM.pptx (56 kb)
Supplementary Table 1. Retention time CVs for technical replicates and the overall biological CV for retention time. (PPTX 55 kb)
13361_2019_2350_MOESM5_ESM.pptx (111 kb)
Supplementary Figure 4. A) Histogram of top 20 proteoforms scaled abundances for all control samples (240 measurements) is approximately normally distributed. B) Q-Q Plot of all proteoforms C) Anderson-Darling test for Normality p-value summary. The vast majority of proteoforms are most likely normally distributed. (PPTX 110 kb)
13361_2019_2350_MOESM6_ESM.pptx (84 kb)
Supplementary Figure 5. Histogram of all p-values from filtered proteoforms between Control and Butyrate treated SUM159 cells. (PPTX 84 kb)
13361_2019_2350_MOESM7_ESM.pptx (558 kb)
Supplementary Figure 6. A) Elution profile of a control SUM159 run that contained early eluting peaks “B” and “C” B) MS1 of early eluting peak “B”. The m/z distributions are similar to those of the predominant H4 proteoforms, but shifted -2 Da C) MS1 of early eluting peak “C”. The m/z distributions are similar to those of the predominant H4 proteoforms, but shifted -18 Da. D) Prosight PC ion map from the most abundant precursor in B. Histone H4 with a -1.027 Da modification is the best fit (p-value and ions matched), indicative of allysine on a C-terminal lysine. Accurate localization was not achieved. E) Prosight PC ion map from the most abundant precursor in C. Histone H4 with a -18 Da modification is the best fit (p-value and ions matched) by Succinimide on D68. (PPTX 558 kb)
13361_2019_2350_MOESM8_ESM.pptx (55 kb)
Supplementary Table 2 Oxidation and sulfate marginalized proteoforms that are significantly increased by butyrate and have the highest fold change. The fully tail acetylated combination “K5acK8acK12acK16ac” is predominantly observed as the most increased. (PPTX 55 kb)
13361_2019_2350_MOESM9_ESM.pptx (56 kb)
Supplementary Table 3 (PPTX 55 kb)

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Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Verna & Marrs McLean Department of Biochemistry & Molecular BiologyBaylor College of MedicineHoustonUSA
  2. 2.Department of Molecular and Cellular BiologyBaylor College of MedicineHoustonUSA

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