Outer Membrane Protease OmpT-Based Strategy for Simplified Analysis of Histone Post-Translational Modifications by Mass Spectrometry

Histone modifications play an important role in regulating transcriptional
gene expression and chromatin processes in eukaryotes. Increasing
researches proved that aberrant post-translational modifications (PTMs)
of histones is associated with many diseases. However, MS-based identification
and quantification of histone PTMs are still challenging. Although
classic chemical derivatization in conjunction with trypsin digestion
is widely used for histone PTMs analysis in a bottom-up strategy,
several side reactions have been observed in practice. In this work,
outer membrane protease T (OmpT) was utilized as a protease for direct
histone proteolysis and generated appropriate lengths of histone peptides
for retention on reversed-phase chromatography. The powerful and unique
tolerance of OmpT for modified lysines and arginines was demonstrated
and can be quantitatively described for the first time, making it
useful for detecting natural modifications. Using the optimized digestion
conditions, we succeeded in identifying 121 histone marks from HEK293T
cells, 42 of which were previously unreported. Additionally, histone
H3 PTMs were quantitatively profiled in the KMS11 multiple myeloma
cells and NSD2 selective knockout KMS11cells, revealing that NSD2
was of high specificity on H3K36 dimethylation. Histone chemical derivatizations
are not required in our strategy, showing a remarkable strength over
the conventional trypsin-based workflow.