Bullet points to evaluate the performance of the middle-down proteomics workflow for histone modification analysis
Mariel Coradin1, Mariel R Mendoza1, Simone Sidoli2
1Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA; Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Methods (San Diego, Calif.)
|February 20, 2020
Summary
Middle-down proteomics improves histone post-translational modification (PTM) analysis by examining longer fragments. This study refines the middle-down workflow, enhancing reproducibility and data quality for PTM identification.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Middle-down proteomics is ideal for studying combinatorial histone post-translational modifications (PTMs).
- The conventional bottom-up approach digests histones into short peptides, while middle-down uses longer polypeptides (50-60 aa) for PTM analysis.
- Middle-down proteomics faces challenges in reproducibility and robustness, hindering quality assessment.
Purpose of the Study:
- To identify critical workflow aspects for evaluating middle-down proteomics experiments.
- To improve the reliability and accuracy of middle-down workflows for histone PTM analysis.
- To provide a streamlined method for assessing middle-down performance.
Main Methods:
- Investigated the impact of porous graphitic carbon (PGC) in desalting steps to prevent sample loss.
- Evaluated different salts in weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) buffers.
- Assessed the effects of salt substitutions (e.g., ethylenediammonium for ammonium) on retention, selectivity, and reproducibility.
Main Results:
- Desalting with C18 material alone caused sample loss; PGC inclusion improved sample recovery.
- Salt choice in WCX-HILIC buffers significantly influenced the retention and selectivity of histone tail fragment variants.
- Ethylenediammonium ions demonstrated marked effects on selectivity and retention compared to ammonium ions.
Conclusions:
- Optimizing desalting and buffer composition is crucial for robust middle-down proteomics.
- The refined workflow enhances the evaluation of middle-down performance for identifying and quantifying histone PTMs.
- This study provides essential benchmarks for assessing the quality of middle-down PTM analyses.
