Chapter Seven – Identification of Posttranslational Modifications of Endogenous Chromatin Proteins From Testicular Cells by Mass Spectrometry

Chromatin architecture in mammalian spermatogenesis undergoes extensive structural and functional reorganization during which several testis-specific histone variants and other chromatin proteins are expressed in a stage-dependent manner. The most dramatic change in chromatin composition is observed during spermiogenesis where nucleosomal chromatin is transformed into nucleoprotamine fiber. Role of posttranslational modification (PTM) of somatic canonical histones and histone variants is well documented and effect several chromatin-templated events. PTM of testis-specific chromatin proteins is proposed to orchestrate chromatin-templated events during mammalian spermatogenesis and their identification and subsequent functional characterization is key to understand chromatin restructuring events and establishment of sperm epigenome. Here, we present protocols for the purification of endogenous testis chromatin proteins from different stages of spermatogenesis and identification of their PTM repertoire by mass spectrometry through examples of testis-specific histone variants (TH2B and HILS1), and transition proteins (TP1 and TP2).