Leveraging unbiased LC-MS analysis to identify histone post-translational modifications associated with age-related decline of learning and memory

Purified histones were washed thrice with the respective solutions in order: 4% perchloric acid, acetone containing 0.2% HCl, and 100% acetone. Precipitated histones were dried using a speed vacuum before being digested for mass spectrometry. Mass spectrometryBottom-up MS/MS was used to elucidate post-translational modifications from the histone samples, as described by Sidoli and colleagues (2016). In brief, dry pellets were resuspended in 50mM NH4HCO3 (pH 8.0), and proteins were subsequently quantified.

Histone derivatisation was carried out using propionic anhydride, followed by trypsin/LysC digestion of the samples. Histone peptide N-termini derivatisation was also carried out using propionic anhydride, after which all the samples were desalted, and run through the Orbitrap Eclipse Tribrid Mass Spectrometer, which made use of a 1 hr gradient (2-38% acetonitrile) as well as an EASY-Spray column. MS data was analyzed using Proteome Discoverer 2.5 with MS Amanda 2.0 and Sequest HT search engines.MS RAW Files legend: Instrument_Date_Histone_aged-conditioned (OC)/young-conditioned (YC)/aged-naive (ON)/young-naive (YN).RAW