Proteomics

Dataset Information

0

Top Down Histone H4 - SUM159 Butyrate


ABSTRACT: Proteins physiologically exist and function as ‘proteoforms’ that arise from one gene but acquire additional function by further variation such as post-translational modification (PTM). When multiple PTMs coexist on single protein molecules top down proteomics becomes the only feasible method; however, most top down methods have limited quantitative capacity and insufficient throughput to truly address proteoform biology. Here we demonstrate that, with meticulous design and diligent consideration of statistics, top down proteomics is 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, including gene expression. 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 online C-3 chromatography, narrowly isolated in MS1 and sequenced with ETD fragmentation. With this method we achieve more than 30 replicates from a single 35mm tissue culture dish by loading 55ng of H4 on column. Parallelization and automation yield a sustained throughput of 12 replicates per day, operating continuously for weeks. 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Mesenchyme Of Mammary Gland, Cell Culture

DISEASE(S): Triple-receptor Negative Breast Cancer

SUBMITTER: Matthew Holt  

LAB HEAD: Nicolas L. Young

PROVIDER: PXD013766 | Pride | 2019-11-22

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
Full_proteoform_allruns.csv Csv
SUM159_But_5mM_4h_H4_1_1.raw Raw
SUM159_But_5mM_4h_H4_1_2.raw Raw
SUM159_But_5mM_4h_H4_1_3.raw Raw
SUM159_But_5mM_4h_H4_2_1.raw Raw
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Publications

High-Throughput Quantitative Top-Down Proteomics: Histone H4.

Holt Matthew V MV   Wang Tao T   Young Nicolas L NL  

Journal of the American Society for Mass Spectrometry 20191118 12


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,  ...[more]

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