Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Nature Protocols volume 13, pages 293–306 (2018)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

References

  1. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    Article  CAS  Google Scholar 

  2. Bantscheff, M., Schirle, M., Sweetman, G., Rick, J. & Kuster, B. Quantitative mass spectrometry in proteomics: a critical review. Anal. Bioanal. Chem. 389, 1017–1031 (2007).

    Article  CAS  Google Scholar 

  3. Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513–2526 (2014).

    Article  CAS  Google Scholar 

  4. Hebert, A.S. et al. Neutron-encoded mass signatures for multiplexed proteome quantification. Nat. Methods 10, 332–334 (2013).

    Article  CAS  Google Scholar 

  5. Ross, P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    Article  CAS  Google Scholar 

  6. Thompson, A. et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904 (2003).

    Article  CAS  Google Scholar 

  7. Hsu, J.L., Huang, S.Y., Shiea, J.T., Huang, W.Y. & Chen, S.H. Beyond quantitative proteomics: signal enhancement of the a1 ion as a mass tag for peptide sequencing using dimethyl labeling. J. Proteome Res. 4, 101–108 (2005).

    Article  CAS  Google Scholar 

  8. Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494 (2009).

    Article  CAS  Google Scholar 

  9. Ong, S.E. & Mann, M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–2660 (2006).

    Article  CAS  Google Scholar 

  10. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  Google Scholar 

  11. Merrill, A.E. et al. NeuCode labels for relative protein quantification. Mol. Cell. Proteomics 13, 2503–2512 (2014).

    Article  CAS  Google Scholar 

  12. Sleno, L. The use of mass defect in modern mass spectrometry. J. Mass Spectrom. 47, 226–236 (2012).

    Article  CAS  Google Scholar 

  13. Gu, S., Pan, S., Bradbury, E.M. & Chen, X. Precise peptide sequencing and protein quantification in the human proteome through in vivo lysine-specific mass tagging. J. Am. Soc. Mass Spectrom. 14, 1–7 (2003).

    Article  CAS  Google Scholar 

  14. Jekel, P.A., Weijer, W.J. & Beintema, J.J. Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis. Anal. Biochem. 134, 347–354 (1983).

    Article  CAS  Google Scholar 

  15. Denisov, E., Damoc, E., Lange, O. & Makarov, A. Orbitrap mass spectrometry with resolving powers above 1,000,000. Int. J. Mass Spectrom. 325–327, 80–85 (2012).

    Article  Google Scholar 

  16. Harsha, H.C., Molina, H. & Pandey, A. Quantitative proteomics using stable isotope labeling with amino acids in cell culture. Nat. Protoc. 3, 505–516 (2008).

    Article  CAS  Google Scholar 

  17. Geiger, T. et al. Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics. Nat. Protoc. 6, 147–157 (2011).

    Article  CAS  Google Scholar 

  18. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  19. Minogue, C.E. et al. Multiplexed quantification for data-independent acquisition. Anal. Chem. 87, 2570–2575 (2015).

    Article  CAS  Google Scholar 

  20. Rhoads, T.W. et al. NeuCode labeling in nematodes: proteomic and phosphoproteomic impact of ascaroside treatment in Caenorhabditis elegans. Mol. Cell. Proteomics 14, 2922–2935 (2015).

    Article  CAS  Google Scholar 

  21. Baughman, J.M. et al. NeuCode proteomics reveals Bap1 regulation of metabolism. Cell Rep. 16, 583–595 (2016).

    Article  CAS  Google Scholar 

  22. Shishkova, E., Hebert, A.S. & Coon, J.J. Now, more than ever, proteomics needs better chromatography. Cell Syst. 3, 321–324 (2016).

    Article  CAS  Google Scholar 

  23. Schütz, W. et al. Extending SILAC to proteomics of plant cell lines. Plant Cell. 23, 1701–1705 (2011).

    Article  Google Scholar 

  24. Kruger, M. et al. SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. Cell 134, 353–364 (2008).

    Article  Google Scholar 

  25. Potts, G.K. et al. Neucode labels for multiplexed, absolute protein quantification. Anal. Chem. 88, 3295–3303 (2016).

    Article  CAS  Google Scholar 

  26. Rhoads, T.W. et al. Neutron-encoded mass signatures for quantitative top-down proteomics. Anal. Chem. 86, 2314–2319 (2014).

    Article  CAS  Google Scholar 

  27. Shortreed, M.R. et al. Elucidating proteoform families from proteoform intact-mass and lysine-count measurements. J. Proteome Res. 15, 1213–1221 (2016).

    Article  CAS  Google Scholar 

  28. Richards, A.L. et al. One-hour proteome analysis in yeast. Nat. Protoc. 10, 701–714 (2015).

    Article  CAS  Google Scholar 

  29. Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705 (2009).

    Article  CAS  Google Scholar 

  30. Gorshkov, M.V., Fornelli, L. & Tsybin, Y.O. Observation of ion coalescence in Orbitrap Fourier transform mass spectrometry. Rapid Commun. Mass Spectrom. 26, 1711–1717 (2012).

    Article  CAS  Google Scholar 

  31. Werner, T. et al. Ion coalescence of neutron encoded TMT 10-plex reporter ions. Anal. Chem. 86, 3594–3601 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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).

Author information

Authors and Affiliations

  1. Genome Center, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Katherine A Overmyer, Alex S Hebert, Michael S Westphall & Joshua J Coon

  2. Morgridge Institute for Research, Madison, Wisconsin, USA

    Katherine A Overmyer & Joshua J Coon

  3. Max Planck Institute of Biochemistry, Martinsried, Germany

    Stefka Tyanova & Jürgen Cox

  4. Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Joshua J Coon

  5. Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Joshua J Coon

Authors
  1. Katherine A Overmyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefka Tyanova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alex S Hebert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael S Westphall
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jürgen Cox
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Joshua J Coon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Ethics declarations

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)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/nprot.2017.121

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.121

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing