It has proven particularly difficult to purify Linker (H1) histones from the model plant Arabidopsis thaliana. This is most likely due to its low nuclear DNA content and the abundance of substances that interfere with protein isolation. These problems have hindered the use of Arabidopsis for in-depth characterization of nuclear proteins by modern techniques based on mass spectrometry (MS). Here, we describe an improved methodology for preparing pure Arabidopsis H1s and separating them by HPLC into fractions corresponding to nonallelic variants. In addition, we outline basic approaches enabling the identification of posttranslational modifications of H1 by MS and their mapping by digestion with different proteases. We also discuss the analysis and interpretation of the acquired data.
Keywords: Arabidopsis thaliana; H1 purification; HPLC; Linker (H1) histones; Mass spectrometry; Posttranslational modifications of H1.