SILAC

SILAC, or stable isotope labeling by amino acids in cell culture, is a quantitative proteomics approach based on metabolic incorporation of non-radioactive, heavy isotopes into cellular proteins. Cells are grown in media containing isotopically labeled essential amino acids, most commonly lysine and arginine, which are incorporated during protein synthesis. After sufficient labeling, proteins from different experimental conditions acquire distinct mass signatures, enabling their relative abundance to be measured by mass spectrometry.

Experimental workflow: two or more cell populations are grown in parallel with light and heavy amino acids (and sometimes a medium isotopic form for three-plex experiments). After harvest, equal amounts of protein are mixed, proteins are digested with proteases such as trypsin, and peptide mixtures are analyzed by LC-MS/MS. The mass spectrometer detects pairs of peptides that differ in mass according to the incorporated isotopes, and software quantifies their intensities to yield relative abundance ratios.

Advantages include accurate, multiplexed quantification with minimal variation from sample handling and compatibility with many proteomic workflows. SILAC can be combined with affinity enrichment or turnover studies and is well suited for comparing transcriptional or signaling states. Limitations include limited applicability to non-dividing cells or primary tissues that cannot be cultured; labeling efficiency and amino acid recycling can affect accuracy; cost and the need for specialized media; and potential biases in extreme experimental conditions.

Variants and related approaches: triple-SILAC uses three isotopic forms to compare more conditions in a single experiment; SILAC can be combined with spike-in standards for absolute quantification. Since its introduction in the early 2000s, SILAC has become a standard method in quantitative proteomics, complementing other labeling strategies such as isobaric tagging and label-free methods.