In eukaryotic cells, DNA is packaged as chromatin into functional units called nucleosomes. These units are composed of an octamer of four core histones (two each of H2A, H2B, H3, and H4)¹^,²^,³^,⁴. Histones are amongst the most abundant and highly conserved proteins in eukaryotes, which are largely responsible for gene regulation⁵. Histone posttranslational modifications (PTMs) play a large role in the regulation of chromatin dynamics and rigger various biological processes such as DNA transcription, replication, and repair⁶. PTMs occur primarily on the accessible surface of the N-terminal regions of histones that are in contact with DNA³^,⁷. However, tail and core modifications influence chromatin structure, altering inter-nucleosome interactions and recruiting specific proteins³^,⁸.

A current challenge during liquid chromatography-mass spectrometry (LC-MS)-based proteomics is the potential co-elution of analytes of interest. In the case of data-dependent analyses (DDA), this translates into the potential loss of several precursor ions during the MS/MS acquisition process⁹. Time-of-flight (ToF) instruments acquire spectra at very high frequency⁹^,¹⁰ (up to tens of kHz)¹¹; this makes them capable of rapidly scanning the total precursor ions within a complex sample (MS1), thus promising optimal sensitivity and MS/MS sequencing rates (up to 100 Hz)⁹ and making them ideal for biological sample analysis¹⁰. Nevertheless, the sensitivity available at these high scan rates is limited by the MS/MS rate⁹. The addition of trapped ion mobility spectrometry (TIMS) in combination with an orthogonal quadrupole time-of-flight (qToF) mass spectrometer was used to mitigate these limitations. In TIMS, all precursor ions are accumulated in tandem and eluted as a function of their mobility, rather than selecting single precursor masses with a quadrupole⁹. Parallel accumulation-serial fragmentation (PASEF) allows for hundreds of MS/MS events per second without any loss of sensitivity⁹.

The principal aim of this work was to show the recent developments of DDA using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation (CID). Histone PTMs were confidently assigned based on their retention times (RTs), mobilities, and fragmentation patterns.

Configuring and Calibrating the TIMS Software Interface for Proteomic Analysis