Metabolic labeling is a relative quantification technique that relies on the replacement of a standard light isotope with a “heavy” isotope (e.g. 15N, 13C, D, or 18O) in cellular proteins. The heavy isotope is incorporated into the protein using a heavy-labeled metabolite that is added to media during cell culture. In a typical metabolic labeling experiment comparing cells grown under two biological conditions, cells under one condition receive heavy-isotope labeled media while cells grown under the other condition receive normal media. Equal numbers of cells from each condition are mixed and processed for mass spectrometric analysis. Proteins from different conditions are distinguished by a known mass shift in their spectra. Relative protein amounts can be quantified using the area under the curve in the extracted ion chromatograms for heavy and light-labeled peptides or by relative fragment ion intensities. The main advantage of metabolic labeling is that the labeled cells and unlabeled cells are mixed as a first step, so proteins have identical losses for all of the proceeding processing steps (). The main disadvantage of metabolic labeling is that it has to be done in tissue culture. Two types of metabolic labeling are used for histone quantification: stable isotope labeling of amino acids in cell culture (SILAC), and labeling of metabolites that become incorporated into the histone modifications.
SILAC was developed by the Mann group in 2002 and since has been used in numerous histone studies (Ong et al., 2002
). It was used recently to investigate partitioning of H3 variants during DNA replication (Xu et al., 2010
). In this study, FLAG epitope-tagged H3.1 and H3.3 variants were grown in HeLa cells. The cells were arrested and switched into media containing 13
-lysine so that all new histones after cell cycle arrest would become labeled. After immunopurification of the tagged variants, and Bottom Up analysis of the immunopurified histones, they discovered that H3.1–H4 tetramers do not split into dimers during replication dependent nucleosome assembly, but H3.3–H4 dimers do split during replication dependent assembly. They present this as support for the hypothesis that histone modifications in newly formed nucleosomes are copied from histones in preexisting nucleosomes.
SILAC can also be used in combination with Top Down mass spectrometry. It was used to study changes in levels of different canonical HeLa cell H2A isoforms across the cell cycle by following the level of incorporation of 13
N labeled valine and arginine into the H2A isoforms after cell synchronization (Boyne et al., 2006
). This study confirmed that the majority of canonical H2A isoforms are produced during S-phase, and showed that levels of different H2A isoforms did not vary much across the cell cycle.
Metabolites can also be used to label specific modifications. Jenuwein and coworkers used 13
-methionine to measure levels of H3K9 methylation after inducing the expression Jmjd2b H3K9 de-methylase in murine cells (Fodor et al., 2006
). Later, the Garcia Lab used heavy-methionine labeling in addition to 13
-lysine labeling to study methylation turnover in site-specific manner and to compare methylation turnover to histone turnover (Zee et al., 2010a
). They found that activating methylation marks such as H3K4 generally turned over faster than repressive marks such as H3K9. They also found that methyl marks had faster turnover than the histones themselves, demonstrating that histones are dynamically modified. In a later paper they demonstrated that PTM turnover between variants was fairly constant and that all histone variants, with the exception of histone H2A variants, turned over at approximately the same rate (Zee et al., 2010b