hSWATH: Unlocking SWATH’s Full Potential for an Untargeted Histone Perspective

Mass
spectrometry (MS) has become the technique of choice for large-scale
analysis of histone post-translational modifications (hPTMs) and their
combinatorial patterns, especially in untargeted settings where novel
discovery-driven hypotheses are being generated. However, MS-based
histone analysis requires a distinct sample preparation, acquisition,
and data analysis workflow when compared to traditional MS-based approaches.
To this end, sequential window acquisition of all theoretical fragment
ion spectra (SWATH) has great potential, as it allows for untargeted
accurate identification and quantification of hPTMs. Here, we present
a complete SWATH workflow specifically adapted for the untargeted
study of histones (hSWATH). We assess its validity on a technical
dataset of time-lapse deacetylation of a commercial histone extract
using HDAC1, which contains a ground truth, i.e., acetylated substrate
peptides reduce in intensity. We successfully apply this workflow
in a biological setting and subsequently investigate the differential
response to HDAC inhibition in different breast cancer cell lines.