Stable Isotope Labeling of Mammals (SILAM).
INTRODUCTIONA general approach in quantitative mass spectrometry is to mix a protein sample containing only natural-abundance isotopes with an identical protein sample containing proteins labeled with heavy stable isotopes (e.g., (2)H, (13)C, (15)N, or (18)O). Introduction of stable isotope labels into proteins alters their molecular weight and such changes can be observed on the mass spectrometer. The relative protein expression is calculated from the ion chromatograms of the labeled and unlabeled peptides. Introduction of stable isotopes via metabolic labeling is an efficient and quantitative method compared to in vitro covalent labeling strategies, because it ensures that every protein is enriched with a heavy stable isotope. Metabolic labeling is routinely performed with cultured cells, ranging from bacteria and yeast to mammalian cells. However, analysis of mammalian tissue allows greater insight into physiology compared to cultured cells. In this protocol, we describe a procedure to metabolically label Rattus norvegicus with (15)N for quantitative mass spectrometry analysis. Stable isotope labeling of mammals (SILAM) allows the global quantitative analysis of any mammalian model of human disease.