TY  - JOUR
TI  - A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
AU  - Bhanu, Natarajan V
AU  - Sidoli, Simone 
AU  - Garcia, Benjamin  A
VL  - 10
IS  - 18
PY  - 2020
DA  - 2020/09/20
SP  - e3756
C1  - Bio-protocol 2020;10:e3756
DO  - 10.21769/BioProtoc.3756
UR  - https://doi.org/10.21769/BioProtoc.3756
AB  - 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.
KW  - Histone
KW  - Post-translational modifications (PTMs)
KW  - Chromatin
KW  - Advantage over Liquid-Chromatography (LC)
KW  - Direct injection
KW  - Mass spectrometry
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False