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 DIA-MS Skyline-based workflow for quantifying histone tail modifications takes advantage of Skyline’s native LFQ 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 co-enriched peptides as iRT anchors.
FAQ
How do I use the Skyline document?
Download the histone_ptm_template.sky.zip file linked below. Open your Skyline software, then open histone_ptm_template.sky.zip and Skyline will unzip it for you. The template contains all the histone PTM targets included in the heavy standard library mix, anchor peptides coenriched in typical histone prep protocols, plus one PRM acquisition of the heavy standard peptides to serve as a chromatogram curation example. You will need Skyline version 20.1 or Skyline-daily to open this document. To import your data, go to File > Import > Results.
How do I get quantitative results?
Skyline can export a quantitative matrix, generated by summing the AUC of each fragment ion marked as "Quantitative" in the document. To export the matrix, go to File > Export > Custom Report, and choose which information you want (e.g. Peptide Modified Sequence, File Name, and Total Area Fragment)
My retention times/fragmentation peaks/stuff looks different from the "reference" data file, what gives?
The peak-picking for co-/closely-eluting isobaric modified peptides is pretty tricky -- this means that you (as the mass spectrometry expert!) will have to do some manual data curation. Skyline's native visualizations will help (e.g. Retention Time - Replicates view, peak area view) but things like dead volume differences across LC systems, column lengths, gradients, and sample matrix changes will cause deviations from the "reference" PRM data included in the document. To get more familiar with how to do manual data curation in Skyline, there are lots of tutorials going through Skyline's various features (https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorials) and webinars (I'd recommend https://skyline.ms/project/home/software/Skyline/events/2015%20Webinars/Webinar%205/begin.view? to get familiar with what to look for when curating quantitative mass spectrometry proteomics data).
What is the difference between the options "total area" or "total area MS1" or "total area fragment"?
All of Skyline's exported "Area" values are calculated as the background-subtracted area-under-the-curve (AUC). Total Area includes the MS1 and MS2 areas (so, precursor and fragment areas); Total Area MS1 is just the MS1/precursor area; Total Area Fragment is just the MS2 area. I always use Total Area Fragment.