Histones form the fundamental unit of the eukaryotic nucleosome consisting of 146 bp of DNA wrapped around protein octamer units. Bottom-up approaches to studying histones result in a mixture of isobaric species, making them challenging to analyze via routine LC-MS/MS. Here, we have utilized trapped ion mobility on a QTOF, to separate isobaric species for the detection of various modforms. Utilizing PASEF workflows, we detect over 2000 peptide sequences and achieve increased coverage of histone modforms without specialized chromatography and data interpretation.
Histones were acid-extracted from Human embryonic kidney 293T cells and derivatized with propionic anhydride/ACN (1:3) pre- and post-digestion with trypsin. Peptides were separated on a 1.6µm C18 25cm x 75µm column (Ionoptiks) using a nanoElute nano LC (Bruker Daltonics) coupled to a trapped ion mobility equipped Q-TOF mass spectrometer (timsTOF Pro). Acquired data were analyzed via Data Analysis (Bruker Daltonics) and PEAKS (Bioinformatics Solutions, Inc) softwares.
Preliminary Data or Plenary Speakers Abstract
Due to the abundance of arginine and lysine amino acids, tryptic digests of histones often produce many fragments of less than 5.Therefore, propionic anhydride is utilized to derivatize the lysine residues resulting in Arg-C like peptides fragments. However, this process coupled with the presence of multiple post translational modifications on histones, produces a complex mixture of isobaric and near-isobaric species. Here, we have utilized standard reverse phase chromatography coupled with PASEF workflows to identify over 200 histone protein groups, 2000 peptide sequences and 6000 peptide spectrum matches (PSM) (at 0.1% FDR as estimated by PEAKS using decoy fusion) from 270ng of protein. Additionally, the orthogonal separation afforded by trapped ion mobility has allowed us to generate more PSMs per modform. This subsequently translates into more modforms identified for a given peptide sequence resulting in improved depth and coverage of PTMs on a particular histone.