Mass spectrometric profiling of histone marks in normal and obstructed kidneys

MS analysis was completed at Active Motif using the Mod Spec^TM platform (Active Motif, Carlsbad, CA, USA). Historically, MS of histones has been problematic. Most protocols use trypsin which cleaves at lysine and arginine residues. However, because of the large number of lysine residues, trypsin digestion produces small peptides that are very hydrophilic and difficult to analyse. To overcome this, lysine residues were converted to propionyl amides so that cleavage occurred at arginine (9). In brief, histones were acid extracted from each sample (n=3 per group), and derivatized via propionylation. A trypsin digestion was then performed, with a second propionic anhydride reaction performed to modify newly formed N-termini, as previously described (9). Samples were then measured in triplicate using the Thermo Scientific TSQ Quantum Ultra mass spectrometer (Thermo Scientific, Waltham, MA, USA) coupled with an UltiMate 3000 Dionex nano-liquid chromatography system (Thermo Scientific). Each value represents the mean relative abundance of each form of a given tryptic peptide from three MS runs. The data was quantified using Skyline (21), and comprises the percent of each modification within the total pool of that amino acid residue (% peptide pool).