Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry

Article metrics


Advancement in proteomics research relies on the development of new, innovative tools for identifying and characterizing proteins. Here, we describe a protocol for analyzing peptides and proteins on a chromatographic timescale by coupling nanoflow reverse-phase (RP) liquid chromatography (LC) to electron-transfer dissociation (ETD) mass spectrometry. For this protocol, proteins can be proteolytically digested before ETD analysis, although digestion is not necessary for all applications. Proteins ≤30 kDa can be analyzed intact, particularly if the objective is protein identification. Peptides or proteins are loaded onto a RP column and are gradient-eluted into an ETD-enabled mass spectrometer. ETD tandem mass spectrometry (MS/MS) provides extensive sequence information required for the unambiguous identification of peptides and proteins and for characterization of posttranslational modifications. ETD is a powerful MS/MS technique and does not compromise the sensitivity and speed necessary for online chromatographic separations. The described procedure for sample preparation, column packing, sample loading and ETD analysis can be implemented in 5–15 h.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Fragmentation scheme for production of ions of type c′ and z′ resulting from the reaction of a fluoranthene radical anion and a multiply protonated peptide6,8.
Figure 2: ETD mass spectrum of the [M + 3H]+3 ion (m/z 519.8) of a phosphorylated peptide derived from the HIV-1 Rev protein.
Figure 3: Diagram of a capillary column utilized for online nanoflow LC analyses.
Figure 4: ETD mass spectra recorded, with and without supplemental activation, on [M + 3H]+3 ions (m/z 823) from residues 18–39 of Adrenocorticotropic Hormone (ACTH 18–39).
Figure 5: ETD/PTR mass spectrum of a 90-residue peptide derived from the Sam68 protein.
Figure 6: ETD/PTR mass spectrum of intact 21-kDa protein, p21.


  1. 1

    Peng, J. & Gygi, S.P. Proteomics: the move to mixtures. J. Mass Spectrom. 36, 1083–1091 (2001).

  2. 2

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

  3. 3

    Yamashita, M. & Fenn, J.B. Electrospray ion source. Another variation on the free-jet theme. J. Phys. Chem. 88, 4451–4459 (1984).

  4. 4

    Eng, J.K., McCormack, A.L., Yates, I. & John, R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

  5. 5

    Geer, L.Y. et al. Open mass spectrometry search algorithm. J. Proteome Res. 3, 958–964 (2004).

  6. 6

    Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanowitz, J. & Hunt, D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 101, 9528–9533 (2004).

  7. 7

    Zubarev, R.A., Kelleher, N.L. & McLafferty, F.W. Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 120, 3265–3266 (1998).

  8. 8

    Coon, J.J. et al. Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 102, 9463–9468 (2005).

  9. 9

    Chi, A., Bai, D.L., Geer, L.Y., Shabanowitz, J. & Hunt, D.F. Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry. Int. J. Mass Spectrom. 259, 197–203 (2007).

  10. 10

    Zubarev, R.A. et al. Electron capture dissociation of gaseous multiply-charged proteins is favored at disulfide bonds and other sites of high hydrogen atom affinity. J. Am. Chem. Soc. 121, 2857–2862 (1999).

  11. 11

    Udeshi, N.D., Shabanowitz, J., Hunt, D.F. & Rose, K.L. Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS. FEBS J. 274, 6269–6276 (2007).

  12. 12

    Taverna, S.D. et al. Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini. Proc. Natl. Acad. Sci. U.S.A. 104, 2086–2091 (2007).

  13. 13

    Chi, A. et al. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 104, 2193–2198 (2007).

  14. 14

    Grigera, P.R. et al. FAK phosphorylation sites mapped by mass spectrometry. J. Cell Sci. 118, 4931–4935 (2005).

  15. 15

    Kjeldsen, F., Giessing, A.M.B., Ingrell, C.R. & Jensen, O.N. Peptide sequencing and characterization of post-translational modifications by enhanced ion-charging and liquid chromatography electron-transfer dissociation tandem mass spectrometry. Anal. Chem. 79, 9243–9252 (2007).

  16. 16

    Swaney, D.L. et al. Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 79, 477–485 (2007).

  17. 17

    Bunger, M.K., Cargile, B.J., Ngunjiri, A., Bundy, J.L. & Stephenson, J.L. Jr. Automated proteomics of E. coli via top-down electron-transfer dissociation mass spectrometry. Anal. Chem. 80, 1459–1467 (2008).

  18. 18

    Martin, S.E., Shabanowitz, J., Hunt, D.F. & Marto, J.A. Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 72, 4266–4274 (2000).

  19. 19

    Schroeder, M.J., Webb, D.J., Shabanowitz, J., Horwitz, A.F. & Hunt, D.F. Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry. J. Proteome Res. 4, 1832–1841 (2005).

  20. 20

    Schroeder, M.J., Shabanowitz, J., Schwartz, J.C., Hunt, D.F. & Coon, J.J. A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal. Chem. 76, 3590–3598 (2004).

  21. 21

    Garcia, B.A., Shabanowitz, J. & Hunt, D.F. Analysis of protein phosphorylation by mass spectrometry. Methods 35, 256–264 (2005).

  22. 22

    Ndassa, Y.M., Orsi, C., Marto, J.A., Chen, S. & Ross, M.M. Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications. J. Proteome Res. 5, 2789–2799 (2006).

  23. 23

    Nuhse, T., Yu, K. & Salomon, A. Unit 18.13 Isolation of Phosphopeptides by Immobilized Metal Ion Affinity Chromatography. In Current Protocols in Molecular Biology (eds. Ausubel, F.M. et al.) John Wiley & Sons, Hoboken, New Jersey 07030 18.13.1–18.13.23.

  24. 24

    Stephenson, J.L. & McLuckey, S.A. Ion/ion reactions in the gas phase: proton transfer reactions involving multiply-charged proteins. J. Am. Chem. Soc. 118, 7390–7397 (1996).

  25. 25

    Pollard, V.W. & Malim, M.H. The HIV-1 Rev protein. Annu. Rev. Microbiol. 52, 491–532 (1998).

  26. 26

    Hammarskjold, M.L. Regulation of retroviral RNA export. Semin. Cell Dev. Biol. 8, 83–90 (1997).

  27. 27

    O'Connor, P.B. et al. Long-lived electron capture dissociation product ions experience radical migration via hydrogen abstraction. J. Am. Soc. Mass Spectrom. 17, 576–585 (2006).

  28. 28

    Lukong, K.E. & Richard, S. Sam68, the KH domain-containing superSTAR. Biochim. Biophys. Acta 1653, 73–86 (2003).

  29. 29

    Coyle, J.H. et al. Sam68 enhances the cytoplasmic utilization of intron-containing RNA and is functionally regulated by the nuclear kinase Sik/BRK. Mol. Cell. Biol. 23, 92–103 (2003).

  30. 30

    Volkmann, N. et al. Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. Science 293, 2456–2459 (2001).

Download references


The authors thank Marie-Louise Hammarskjold, David Rekosh, Yukiko Misawa and Emily Sloan for providing the Sam68 and Rev protein samples and Dorothy Schafer and Tatyana Kotova for providing the Arp2/3 protein sample. This work was supported by grants from the National Institutes of Health (GM37537 to D.F. Hunt).

Author information

Correspondence to Donald F Hunt or Kristie L Rose.

Rights and permissions

Reprints and Permissions

About this article

Further reading


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.