Top-down proteomics is an emerging mass spectrometric technique with the ability to identify intact proteoforms. Currently, there is no method to concurrently quantify proteins and post-translational modifications (PTMs) through top-down methods. Histones have a diverse range of proteoforms stemming from their tails which are susceptible to chemical modifications and directly influence the compaction of the chromatin. Due to the positively-charged and unstructured termini, quantification of histone PTMs remains a challenge for bottom-up histone analysis. The typical analysis relies on propionylation that allows proteolysis to only occur at the C-terminal of arginine residues. The peptides then require assembly and reconstruction to provide information on the histone and this can produce ambiguous results. Top-down proteomics, where intact proteoforms are analysed is therefore a better fit for quantifying multiple PTMs on histone isoforms. We have developed a process to investigate the quantitative changes of PTMs on histones grown in varying media conditions.
Histones grown in HeLa cells were acid extracted and dissolved in milliQ H2O and 1% FA and 2 µg of each sample was loaded onto a C8 HPLC column and subjected to on-line fractionation coupled to a 15-Tesla Solarix FT-ICR mass spectrometer. Raw files were exported and subjected to an in-house developed program where theoretical intact proteoform masses are matched to the deconvoluted LC-MS data and the relative quantification of the proteoforms are extracted. Current results show significant differences in patterns of PTMs found for histones grown in HeLa cells in media with and without amino acids.
The development of quantifying algorithms for high resolution top-down proteomics will determine the magnitude of different PTMs that occur simultaneously. Here, the identification and quantification of concurrent PTMs on histone isoforms will add great value to the knowledge of histone mechanisms.