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


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.

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Figure 1: Lysine isotopologs and theoretical resolution calculations.
Figure 2: Scan sequences for NeuCode 3-plex analysis.
Figure 3: Commercially available lysine isotopologs.
Figure 4: Multiplexing options with NeuCode labels.
Figure 5: Suggested workflow for NeuCode metabolic labeling.
Figure 6: Example plots from MaxQuant's 'Viewer' with 3-plex and 2-plex NeuCode samples.


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We are grateful to M. Rush for comments during the writing process. This work was supported by the National Institutes of Health (P41 GM108538). K.A.O. gratefully acknowledges the support from a US National Library of Medicine training grant (5T15LM007359).

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Authors and Affiliations



S.T. and J.C. developed the MaxQuant analysis tool. K.A.O., A.S.H., and M.S.W. analyzed the data. K.A.O., A.S.H., J.C., and J.J.C. wrote the manuscript.

Corresponding author

Correspondence to Joshua J Coon.

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Competing interests

A.S.H. and J.J.C. are co-inventors on a patent application (US 13/660677) related in part to the material presented here. The other authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 High-abundance ion populations in the Orbitrap can exhibit space charge–induced frequency shifts, resulting in coalescence of the observed m/z values.

Coalescence is more likely to occur with NeuCode peaks due to the close mass-to-charge but will occur in proportion to the abundance of the ion population. Exampled in this figure is the observed coalescence of the NeuCode channels with increased ion intensity. Reduction of the automatic gain control (AGC) target will limit the ions permitted into the Orbitrap mass analyzer and thus minimize the coalescence of NeuCode peaks (see ?Troubleshooting).

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 and Supplementary Tables 1 and 2. (PDF 351 kb)

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Overmyer, K., Tyanova, S., Hebert, A. et al. Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice. Nat Protoc 13, 293–306 (2018).

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