Abstract
Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.
Original language | English (US) |
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Article number | e3756 |
Journal | Bio-protocol |
Volume | 10 |
Issue number | 18 |
DOIs | |
State | Published - 2020 |
Keywords
- Advantage over Liquid-Chromatography (LC)
- Chromatin
- Direct injection
- Histone
- Mass spectrometry
- Post-translational modifications (PTMs)
ASJC Scopus subject areas
- General Neuroscience
- General Biochemistry, Genetics and Molecular Biology
- General Immunology and Microbiology
- Plant Science