A Super-SILAC Approach for Profiling Histone Posttranslational Modifications

Abstract

Histone posttranslational modifications (PTMs) play an important role in the regulation of gene expression and have been implicated in a multitude of physiological and pathological processes. During the last decade, mass spectrometry (MS) has emerged as the most accurate and versatile tool to quantitate histone PTMs. Stable-isotope labeling by amino acids in cell culture (SILAC) is an MS-based quantitation strategy involving metabolic labeling of cells, which has been applied to global protein profiling as well as histone PTM analysis. The classical SILAC approach is associated with reduced experimental variability and high quantitation accuracy, but provides limited multiplexing capabilities and can be applied only to actively dividing cells, thus excluding clinical samples. Both limitations are overcome by an evolution of classical SILAC involving the use of a mix of heavy-labeled cell lines as a spike-in standard, known as "super-SILAC". In this chapter, we will provide a detailed description of the optimized protocol used in our laboratory to generate a histone-focused super-SILAC mix and employ it as an internal standard for histone PTM quantitation.

Keywords: Epigenetics; Histone posttranslational modifications; Mass spectrometry; Super-SILAC.