A histone PTM Skyline template with spectral library and iRT calculator

Despite a growing interest in epigenetics, performing proteomics studies of histone tail marks remains highly specialized. Mass spectrometry of histone tail marks is difficult due to the variety of modifications, coeluting isoforms, and dynamic range. Conventionally, these challenges have been met with database searching shotgun DDA, which detects histone marks but doesn’t provide accurate quantification; or optimizing high-level PRM methods, which accurately quantifies but cannot detect novel histone marks. Here, we have designed a robust histone DIA method and a flexible Skyline-based analysis workflow to more accurately and precisely quantify histone marks. Our targeted mass spectrometry Skyline-based workflow for quantifying histone tail modifications takes advantage of Skyline’s latest features, including staggered DIA isolation window demultiplexing to process raw data, the “Quantitative” fragment ion demarcation for site-localizing isobaric histone marks, and a retention time calculator that uses peptides co-enriched in the histone preparation as iRT anchors.