A variety of different posttranslational modifications (PTMs) on histone proteins display an important layer of chromatin regulation. PTMs mostly occur on the N-terminal tails of histones that are protruding out of the nucleosome, with histones H3 and H4 featuring more described PTMs than H2A and H2B (Figure 1). Among the set of known histone PTMs, methylation and acetylation are the most abundant. In order to understand more about chromatin regulation, it is crucial to analyze histone modifications in a quantitative way.
Different technical approaches are available to determine the amount and location of these histone modifications, with antibody-based techniques and mass spectrometry being the most popular. We will describe a procedure to isolate histones, prepare them for MS analysis and interpret the results (Peters et al, 2003).
Due to their basicity, histones can be isolated from biological tissues by extracting with hydrochloric acid (HCl) (Villar-Garea et al, 2008). Further separation by SDS-PAGE gel and Coomassie staining will show a characteristic pattern of histones (Figure 2). The corresponding bands can be excised from the gel to undergo protease digestion prior to MS analysis. Commonly, trypsin is used to cleave proteins
after lysine and arginine. As these amino acids are very abundant in histone tails, trypsin will generate peptides too short to be analyzed efficiently by MS. Therefore, a treatment with propionic anhydride is performed to block lysines from tryptic cleavage. By that means, longer peptides are generated after tryptic digestion and can be better analyzed in subsequent steps (Figure 3).
The resulting peptides are then separated by C18 reversed phase chromatography (RP-C18). The eluting peptides are directly ionized and infused into the mass spectrometer, which measures the mass-to-charge ratio (m/z) of these infused ions. Additionally, the most intense ions present at a certain time point are isolated and collided with an inert gas, generating fragment ions recorded in a so-called MS/MS spectrum. This spectrum contains additional information that together with the m/z value will be used to identify the peptides and the location and mass of PTMs present.
Munich Center of Integrated Protein Science (CIPSM) and Adolf-Butenandt-Institut, Schillerstr. 44, 80336 München, Germany
Corresponding author: Ignasi Forne, Teresa Barth