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High-Throughput Quantitative Top-Down Proteomics: Histone H4
- Source :
- Journal of the American Society for Mass Spectrometry. 30:2548-2560
- Publication Year :
- 2019
- Publisher :
- American Chemical Society (ACS), 2019.
-
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.
- Subjects :
- Proteomics
Protein molecules
010401 analytical chemistry
Sodium butyrate
Computational biology
010402 general chemistry
Top-down proteomics
01 natural sciences
Cell Line
0104 chemical sciences
Histones
Histone H4
chemistry.chemical_compound
chemistry
Tandem Mass Spectrometry
Structural Biology
Protein purification
Humans
Protein Processing, Post-Translational
Throughput (business)
Chromatography, High Pressure Liquid
Spectroscopy
Function (biology)
Subjects
Details
- ISSN :
- 10440305
- Volume :
- 30
- Database :
- OpenAIRE
- Journal :
- Journal of the American Society for Mass Spectrometry
- Accession number :
- edsair.doi.dedup.....b611b3322cd8c67d5b7e0afb84053e15