Skip to main content
SpringerLink
Log in

Mass Spectral Enhanced Detection of Ubls Using SWATH Acquisition: MEDUSA—Simultaneous Quantification of SUMO and Ubiquitin-Derived Isopeptides

  • Research Article
  • Published:
Journal of The American Society for Mass Spectrometry

Abstract

Protein modification by ubiquitination and SUMOylation occur throughout the cell and are responsible for numerous cellular functions such as apoptosis, DNA replication and repair, and gene transcription. Current methods for the identification of such modifications using mass spectrometry predominantly rely upon tryptic isopeptide tag generation followed by database searching with in vitro genetic mutation of SUMO routinely required. We have recently described a novel approach to ubiquitin and SUMO modification detection based upon the diagnostic a′ and b′ ions released from the isopeptide tags upon collision-induced dissociation of reductively methylated Ubl isopeptides (RUbI) using formaldehyde. Here, we significantly extend those studies by combining data-independent acquisition (DIA) with alternative labeling reagents to improve diagnostic ion coverage and enable relative quantification of modified peptides from both MS and MS/MS signals. Model synthetic ubiquitin and SUMO-derived isopeptides were labeled with mTRAQ reagents (Δ0, Δ4, and Δ8) and subjected to LC-MS/MS with SWATH acquisition. Novel diagnostic ions were generated upon CID, which facilitated the selective detection of these modified peptides. Simultaneous MS-based and MS/MS-based relative quantification was demonstrated for both Ub and SUMO-derived isopeptides across three channels in a background of mTRAQ-labeled Escherichia coli digest.

ᅟ

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Finley, D., Chau, V.: Ubiquitination. Annu. Rev. Cell Biol. 7, 25–69 (1991)

    Article  CAS  Google Scholar 

  2. Hay, R.T.: SUMO: a history of modification. Mol. Cell 18, 1–12 (2005)

    Article  CAS  Google Scholar 

  3. Mukhopadhyay, D., Riezman, H.: Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315, 201–205 (2007)

    Article  CAS  Google Scholar 

  4. Bergink, S., Jentsch, S.: Principles of ubiquitin and SUMO modifications in DNA repair. Nature 458, 461–467 (2009)

    Article  CAS  Google Scholar 

  5. Hochstrasser, M.: Origin and function of ubiquitin-like proteins. Nature 458, 422–429 (2009)

    Article  CAS  Google Scholar 

  6. Baek, S.H.: A novel link between SUMO modification and cancer metastasis. Cell Cycle 5, 1492–1495 (2006)

    Article  CAS  Google Scholar 

  7. Sun, Y.: E3 ubiquitin ligases as cancer targets and biomarkers. Neoplasia 8, 645–654 (2006)

    Article  CAS  Google Scholar 

  8. Denuc, A., Marfany, G.: SUMO and ubiquitin paths converge. Biochem. Soc. Trans. 38, 34–39 (2010)

    Article  CAS  Google Scholar 

  9. Hunter, T., Sun, H.: Crosstalk between the SUMO and ubiquitin pathways. Ernst Schering Found. Symp. Proc. 1, 1–16 (2008)

    Article  CAS  Google Scholar 

  10. Yang, X.J.: Multisite protein modification and intracellular signaling. Oncogene 24, 1653–1662 (2005)

    Article  CAS  Google Scholar 

  11. Peng, J., Schwartz, D., Elias, J.E., Thoreen, C.C., Cheng, D., Marsischky, G., Roelofs, J., Finley, D., Gygi, S.P.: A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 8, 921–926 (2003)

    Article  CAS  Google Scholar 

  12. Nielsen, M.L., Michiel Vermeulen, M., Tiziana Bonaldi, T., Cox, J., Moroder, L., Mann, M.: Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat. Methods 5, 459–460 (2008)

    Article  CAS  Google Scholar 

  13. Warren, M.R.E., Parker, C.E., Mocanu, V., Klapper, D., Borchers, C.E.: Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic product ions for protein ubiquitination. Rapid Commun. Mass Spectrom. 19, 429–437 (2005)

    Article  CAS  Google Scholar 

  14. Chicooree, N., Connolly, Y., Tan, C.-T., Malliri, A., Li, Y., Smith, D.L., Griffiths, J.R.: Enhanced detection of ubiquitin isopeptides using reductive methylation. J. Am. Soc. Mass Spectrom. 24, 421–430 (2013)

    Article  CAS  Google Scholar 

  15. Hsu, J.L., Huang, S.Y., Chow, N.H., Chen, S.H.: Stable isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 75, 6843–6852 (2003)

    Article  CAS  Google Scholar 

  16. Lamoliatte, F., Bonneil, E., Durette, C., Caron-Lizotte, O., Wildemann, D., Zerweck, J., Wenshuk, H., Thibault, P.: Targeted identification of SUMOylation sites in human proteins using affinity enrichment and Paralog-specific reporter ions. Mol. Cell Proteomics 12, 2536–2550 (2013)

    Article  CAS  Google Scholar 

  17. Cooper, H.J., Tatham, M.H., Jaffray, E., Heath, J.K., Lam, T.T., Marshall, A.G., Hay, R.T.: Fourier transform ion cyclotron resonance mass spectrometry for the analysis of small ubiquitin-like modifier (SUMO) modification: identification of lysines in RanBP2 and SUMO targeted for modification during the E3 AutoSUMOylation reaction. Anal. Chem. 77, 6310–6319 (2005)

    Article  CAS  Google Scholar 

  18. Chicooree, N., Griffiths, J.R., Connolly, Y., Tan, C.-T., Malliri, A., Eyers, C.A., Smith, D.L.: A novel approach to the analysis of SUMOylation with the independent use of trypsin and elastase digestion followed by database searching utilising consecutive residue addition to lysine. Rapid Commun. Mass Spectrom. 27, 127–134 (2013)

    Article  CAS  Google Scholar 

  19. Chicooree, N., Griffiths, J.R., Connolly, Y., Smith, D.L.: Chemically facilitating the generation of diagnostic ions from SUMO(2/3) remnant isopeptides. Rapid Commun. Mass Spectrom. 27, 2108–2114 (2013)

    Article  CAS  Google Scholar 

  20. 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 

  21. DeSouza, L.V., Taylor, A.M., Li, W., Minkoff, M.S., Romaschin, A.D., Colgan, T.J., Siu, K.W.M.: Multiple reaction monitoring of mTRAQ-labeled peptides enables absolute quantification of endogenous levels of a potential cancer marker in cancerous and normal endometrial tissues. J. Proteome Res. 7, 3525–3534 (2008)

    Article  CAS  Google Scholar 

  22. Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A., Pappin, D.J.: Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell Proteomics 3, 1154–1169 (2004)

    Article  CAS  Google Scholar 

  23. Heumann, K.G.: Isotope dilution mass spectrometry. Int. J. Mass Spectrom. Ion Process. 118/119, 575–592 (1992)

    Article  Google Scholar 

  24. Roepstorff, P., Fohlman, J.: Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed. Mass Spectrom. 11, 601 (1984)

    Article  CAS  Google Scholar 

  25. Carr, S.A., Huddleston, M.J., Annan, R.S.: Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry. Anal. Biochem. 239, 180–192 (1996)

    Article  CAS  Google Scholar 

  26. Mann, M., Hendrickson, R.C., Pandey, A.: Analysis of proteins and proteomes by mass spectrometry. Annu. Rev. Biochem. 70, 437–473 (2001)

    Article  CAS  Google Scholar 

  27. Domon, B., Aebersold, R.: Mass spectrometry and protein analysis. Science 312, 212–217 (2006)

    Article  CAS  Google Scholar 

  28. Plumb, R.S., Johnson, K.A., Rainville, P., Smith, B.W., Wilson, I.D., Castro-Perez, J.M., Nicholson, J.K.: UPLC/MSE; a new approach for generating molecular fragment information for biomarker structure elucidation. Rapid Commun. Mass Spectrom. 20, 1989–1994 (2006)

    Article  CAS  Google Scholar 

  29. Geiger, T., Cox, J., Mann, M.: Proteomics on an Orbitrap benchtop mass spectrometer using all-ion fragmentation. Mol. Cell Proteomics 9, 2252–2261 (2010)

    Article  CAS  Google Scholar 

  30. Gillet, L.C., Navarro, P., Tate, S., Rost, H., Selevsek, N., Reiter, L., Bonner, R., Aebersold, R.: Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol. Cell Proteomics 11, (2012). doi:10.1074/mcp.0111.016717

  31. Silva, J.C., Denny, R., Dorschel, C., Gorenstein, M.V., Li, G.Z., Richardson, K., Wall, D., Geromanos, S.J.: Simultaneous qualitative and quantitative analysis of the Escherichia coli proteome: a sweet tale. Mol. Cell Proteomics 5, 589–607 (2006)

    Article  CAS  Google Scholar 

  32. Shi, Y., Bajrami, B., Morton, M., Yao, X.: Cyclophosphoramidate ion as mass defect marker for efficient detection of protein serine phosphorylation. Anal. Chem. 80, 7614–7623 (2008)

    Article  CAS  Google Scholar 

  33. Zappacosta, F., Collingwood, T.S., Huddlestone, M.J., Annan, R.S.: A quantitative results-driven approach to analyzing multisite protein phosphorylation. Mol. Cell Proteomics 5, 2019–2030 (2006)

    Article  CAS  Google Scholar 

  34. March, R.E.: An introduction to quadrupole ion trap mass spectrometry. J. Mass Spectrom. 32, 351–369 (1997)

    Article  CAS  Google Scholar 

Download references

Acknowledgments

N.C. thanks the EPSRC for financial support. Y.C, J.G, and D.S were funded by Cancer Research, UK. The authors thank Dr. Christie Hunter for valuable advice on using SWATH acquisition, and Craig Mageean for assistance with figure generation.

Author information

Authors and Affiliations

  1. CRUK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK

    John R. Griffiths, Navin Chicooree, Yvonne Connolly & Duncan L. Smith

  2. School of Chemistry, University of Manchester, Manchester, M13 9SU, UK

    Navin Chicooree

  3. AB SCIEX, Phoenix House, Warrington, WA1 1RX, UK

    Milla Neffling, Catherine S. Lane & Thomas Knapman

Authors
  1. John R. Griffiths
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Navin Chicooree
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yvonne Connolly
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Milla Neffling
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Catherine S. Lane
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas Knapman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Duncan L. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John R. Griffiths.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1

(DOCX 36 kb)

Table S2

(DOCX 51 kb)

Table S3

(DOCX 55 kb)

Table S4

(DOCX 55 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Griffiths, J.R., Chicooree, N., Connolly, Y. et al. Mass Spectral Enhanced Detection of Ubls Using SWATH Acquisition: MEDUSA—Simultaneous Quantification of SUMO and Ubiquitin-Derived Isopeptides. J. Am. Soc. Mass Spectrom. 25, 767–777 (2014). https://doi.org/10.1007/s13361-014-0835-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13361-014-0835-x

Key words

Search

Navigation