Multi-plexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice

摘要

We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM)). This method currently enables simultaneous comparison of up to 9 treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in neutron binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologues of lysine are introduced to cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is approximately two weeks for cultured cells and 3-4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During MS data acquisition, high resolution MS¹ spectra (≥240,000 resolving power @ m/z 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time is 3-5 weeks.