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

Nat Protoc. 2018 Jan;13(1):293-306. doi: 10.1038/nprot.2017.121. Epub 2018 Jan 11.


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 nine 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 the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 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 mass spectrometry (MS) data acquisition, high-resolution MS1 spectra (≥240,000 resolving power at 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 for the protocol is 3-5 weeks.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids
  • Animals
  • Cells, Cultured
  • Humans
  • Isotope Labeling / methods*
  • Lysine / metabolism
  • Lysine / radiation effects
  • Mice
  • Neutrons
  • Peptides
  • Proteome / analysis
  • Proteomics / methods*
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Serine Endopeptidases
  • Software
  • Tandem Mass Spectrometry


  • Amino Acids
  • Peptides
  • Proteome
  • Saccharomyces cerevisiae Proteins
  • Serine Endopeptidases
  • lysyl endopeptidase
  • Lysine