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

Histone extraction from mouse hippocampi was carried out using a column-based extraction protocol following manufacturer’s recommendations (Active Motif, #40025). Briefly, nuclei were first isolated by rinsing hippocampi in ice-cold nuclei extraction buffer, followed by mincing into <1 mm pieces and transferred to a homogenizer. Tissue preparations were then left overnight in the nuclei isolation buffer to extract crude histones. Crude histones were then neutralized, and samples were transferred into a resin- column for isolation of histones. Columns were washed with a series of washing buffers provided by the kit. Histones were then eluted, purified, and precipitated by adding perchloric acid at a final concentration of 4%. 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