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.

  • Article
  • Published:

Informatics and multiplexing of intact protein identification in bacteria and the archaea

Nature Biotechnology volume 19, pages 952–957 (2001)Cite this article

Abstract

Although direct fragmentation of protein ions in a mass spectrometer is far more efficient than exhaustive mapping of 1–3 kDa peptides for complete characterization of primary structures predicted from sequenced genomes, the development of this approach is still in its infancy. Here we describe a statistical model (good to within ∼5%) that shows that the database search specificity of this method requires only three of four fragment ions to match (at ±0.1 Da) for a 99.8% probability of being correct in a database of 5,000 protein forms. Software developed for automated processing of protein ion fragmentation data and for probability-based retrieval of whole proteins is illustrated by identification of 18 archaeal and bacterial proteins with simultaneous mass-spectrometric (MS) mapping of their entire primary structures. Dissociation of two or three proteins at once for such identifications in parallel is also demonstrated, along with retention and exact localization of a phosphorylated serine residue through the fragmentation process. These conceptual and technical advances should assist future processing of whole proteins in a higher throughput format for more robust detection of co- and post-translational modifications.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Number of protein forms in a database vs. number of input fragment ion masses for a 1% chance of spuriously matching an incorrect protein.
Figure 2: An illustration of direct MS/MS for the analysis of intact protein mixtures.
Figure 3: Simultaneous dissociation of the two intact proteins of Figure 2A; 9.4 T, 100 scans (2 s/scan).
Figure 4: Illustration of protein identification from a mixture with simultaneous characterization of a post-translational modification.

Similar content being viewed by others

References

  1. Mann, M. & Wilm, M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390–4399 (1994).

    Article  CAS  Google Scholar 

  2. Yates, J.R., Eng, J.K., Clauser, K.R. & Burlingame, A.L. Search of sequence databases with uninterpreted high-energy collision-induced dissociation spectra of peptides. J. Am. Soc. Mass Spectrom. 7, 1089–1098 (1996).

    Article  CAS  Google Scholar 

  3. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  Google Scholar 

  4. Fountoulakis, M., Takacs, M.F., Berndt, P., Langen, H. & Takacs, B. Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography. Electrophoresis 20, 2181–2195 (1999).

    Article  CAS  Google Scholar 

  5. Washburn, M.P., Wolters, D. & Yates, J. R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 (2001).

    Article  CAS  Google Scholar 

  6. Regula, J.T. et al. Towards a two-dimensional proteome map of Mycoplasma pneumoniae. Electrophoresis 21, 3765–3780 (2000).

    Article  CAS  Google Scholar 

  7. Wilkins, M.R. et al. High-throughput mass spectrometric discovery of protein post-translational modifications. J. Mol. Biol. 289, 645–657 (1999).

    Article  CAS  Google Scholar 

  8. Oda, Y., Nagasu, T. & Chait, B.T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379–382 (2001).

    Article  CAS  Google Scholar 

  9. Zhou, H., Watts, J.D. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378 (2001).

    Article  CAS  Google Scholar 

  10. Mann, M. Quantitative proteomics? Nat. Biotechnol. 17, 954–955 (1999).

    Article  CAS  Google Scholar 

  11. Kelleher, N.L. et al. Top down versus bottom up protein characterization by tandem high-resolution mass spectrometry. J. Am. Chem. Soc. 121, 806–812 (1999).

    Article  CAS  Google Scholar 

  12. Reiber, D.C., Grover, T.A. & Brown, R.S. Identifying proteins using matrix-assisted laser desorption/ionization in-source fragmentation data combined with database searching. Anal. Chem. 70, 673–683 (1998).

    Article  CAS  Google Scholar 

  13. Cargile, B., McLuckey, S. & Stephenson, J. Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions. Anal. Chem. 73, 1277–1285 (2001).

    Article  CAS  Google Scholar 

  14. Chong, B. et al. Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis. Anal. Chem. 73, 1219–1227 (2001).

    Article  CAS  Google Scholar 

  15. Kelleher, N.L. et al. Efficient sequence analysis of the six gene products (7–74 kDa) from the Escherichia coli thiamine biosynthetic operon by tandem high-resolution mass spectrometry. Protein Sci. 7, 1796–1801 (1998).

    Article  CAS  Google Scholar 

  16. Forbes, A.J., Mazur, M.T. & Kelleher, N.L. Toward efficient analysis of >70 kDa proteins with 100% sequence coverage. Eur. Mass Spectrom. 7, in press (2001).

  17. Mørtz, E. et al. Sequence tag identification of intact proteins by matching tandem mass spectral data against sequence databases. Proc. Natl. Acad. Sci. USA 93, 8264–8267 (1996).

    Article  Google Scholar 

  18. Kelleher, N.L., Costello, C.A., Begley, T.P. & McLafferty, F.W. Thiaminase I (42 kDa) heterogeneity, sequence refinement, and active site location from high-resolution tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 6, 981–984 (1995).

    Article  CAS  Google Scholar 

  19. Li, W., Hendrickson, C.L., Emmett, M.R. & Marshall, A.G. Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 71, 4397–4402 (1999).

    Article  CAS  Google Scholar 

  20. Perkins, D., Pappin, D., Creasy, D. & Cottrell, J. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    Article  CAS  Google Scholar 

  21. Zhang, W. & Chait, B. ProFound: an expert system for protein identification using mass spectrometric peptide mapping information. Anal. Chem. 72, 2482–2489 (2000).

    Article  CAS  Google Scholar 

  22. Masselon, C. et al. Accurate mass multiplexed tandem mass spectrometry for high-throughput polypeptide identification from mixtures. Anal. Chem. 72, 1918–1924 (2000).

    Article  CAS  Google Scholar 

  23. Senko, M.W., Beu, S.C. & McLafferty, F.W. Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions. J. Am. Soc. Mass Spectrom. 6, 229–233 (1995).

    Article  CAS  Google Scholar 

  24. Schaaff, G.T., Cargille, B., Stephenson, J.L.J., & McLuckey, S.A. Ion trap collisional activation of the (M + 2H)2+–(M + 17H)17+ ions of human hemoglobin b-chain. Anal. Chem. 72, 899–907 (2000).

    Article  CAS  Google Scholar 

  25. Horn, D., Ge, Y. & McLafferty, F. Activated ion electron capture dissociation for mass spectral sequencing of larger (42 kDa) proteins. Anal. Chem. 72, 4778–4784 (2000).

    Article  CAS  Google Scholar 

  26. Mukhopadhyay, B., Johnson, E.F. & Wolfe, R.S. A novel pH2 control on the expression of flagella in the hyperthermophilic strictly hydrogenotrophic methanarchaeaon Methanococcus jannaschii. Proc. Natl. Acad. Sci. USA 97, 11522–11527 (2000).

    Article  CAS  Google Scholar 

  27. http://www.ncifcrf.gov/RESID/ RESID and NRL-3D databases.

  28. Napper, S. et al. Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inactivation by phosphorylation of serine-46 in HPrs from gram-positive bacteria. Biochemistry 35, 11260–11267 (1996).

    Article  CAS  Google Scholar 

  29. Neubauer, G. & Mann, M. Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations. Anal. Chem. 71, 235–242 (1999).

    Article  CAS  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. Kelleher, N.L. et al. Localization of labile posttranslational modifications by electron capture dissociation: the case of g-carboxyglutamic acid. Anal. Chem. 71, 4250–4253 (1999).

    Article  CAS  Google Scholar 

  32. Belov, M.E. et al. Design and performance of an ESI interface for selective external ion accumulation coupled to a Fourier transform ion cyclotron mass spectrometer. Anal. Chem. 73, 253–261 (2001).

    Article  CAS  Google Scholar 

  33. Oda, Y., Huang, K., Cross, F.R., Cowburn, D. & Chait, B.J. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591–6596 (1999).

    Article  CAS  Google Scholar 

  34. Mukhopadhyay, B., Johnson, E.F. & Wolfe, R.S. Reactor-scale cultivation of the hyperthermophilic methanarchaeon Methanococcus jannaschii to high cell densities. Appl. Environ. Microbiol. 65, 5059–5065 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Taylor, P. Recovery of human Mycoplasmas. In Mycoplasma protocols. (eds Miles, R. & Nicholas, R.) 25–35 (Humana Press, Totowa, NJ; 1998).

    Chapter  Google Scholar 

  36. Emmett, M.R. & Caprioli, R.M. Micro-electrospray mass spectrometry: ultra-high-sensitivity analysis of peptides and proteins. J. Am. Soc. Mass Spectrom. 5, 605–613 (1994).

    Article  CAS  Google Scholar 

  37. Kofel, P., Allemann, M., Kellerhals, H.P. & Wanczek, K.P. External trapped ion-source for ion cyclotron resonance spectrometry. Int. J. Mass Spectrom. Ion Processes 87, 237–247 (1989).

    Article  CAS  Google Scholar 

  38. Senko, M.W., Hendrickson, C.L., Emmett, M.R., Shi, S.D.-H. & Marshall, A.G. External accumulation of ions for enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. J. Am. Soc. Mass Spectrom. 8, 970–976 (1997).

    Article  CAS  Google Scholar 

  39. Horn, D.M., Zubarev, R.A. & McLafferty, F.W. Automated reduction and interpretation of high-resolution electrospray mass spectra of large molecules. J. Am. Soc. Mass Spectrom. 11, 320–332 (2000).

    Article  CAS  Google Scholar 

  40. Loo, J.A., Edmonds, C.G. & Smith, R.D. Tandem mass spectrometry of very large molecules: serum albumin sequence information from multiply charged ions formed by electrospray ionization. Anal. Chem. 63, 2488–2499 (1991).

    Article  CAS  Google Scholar 

  41. Little, D.P., Speir, J.P., Senko, M.W., O' Connor, P.B. & McLafferty, F.W. Infrared multiphoton dissociation of large multiply charged ions for biomolecule sequencing. Anal. Chem. 66, 2809–2815 (1994).

    Article  CAS  Google Scholar 

  42. Gauthier, J.W., Trautman, T.R. & Jacobson, D.B. Sustained off-resonance irradiation for collision-activated dissociation involving Fourier-transform mass-spectrometery-collision-activated dissociation technique that emulates infrared multiphoton dissociation. Anal. Chim. Acta 246, 211–225 (1991).

    Article  CAS  Google Scholar 

  43. Senko, M.W., Canterbury, J.D., Guan, S. & Marshall, A.G. A high-performance modular data system for Fourier transform ion cyclotron resonance mass spectrometry. Rap. Commun. Mass Spectrom. 10, 1839–1844 (1996).

    Article  CAS  Google Scholar 

  44. http://www.zmbh.uni-heidelberg.de/M_pneumoniae/ Richard Hermann's group. The Mycoplasma pneumoniae genome project.

Download references

Acknowledgements

The authors thank Biswarup Mukhopadhyay (supported by the Department of Energy grant DE-FG02-87ER13651 and National Institutes of Health grant GM 51334 to Ralph S. Wolfe) for a gift of M. jannaschii cells, Gary Olsen for his M. jannaschii database, and Jason Bucholtz and Tim Murphy for computer assistance. We also thank John Quinn, Mark Emmett, Chris Hendrickson, and Alan Marshall for support with 9.4 T instrumentation (National Science Foundation; National High-Field FT-ICR MS facility, NHMFL, NSF CHE-94-13008). N.L.K. received support from a University of Illinois Critical Research Initiative, a NIH K22 Award (AI 01748-01), the Camille and Henry Dreyfus New Faculty Awards Program, and the Burroughs-Wellcome and Searle Foundations. B.J.C. received a NSF graduate fellowship. We also thank John Garavelli for informative discussions and David Horn and Greg Blakeley for help in streamlining data analysis software.

Author information

Author notes

  1. Fanyu Meng and Benjamin J. Cargile: These two authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, 61801, IL

    Fanyu Meng, Benjamin J. Cargile, Leah M. Miller, Andrew J. Forbes, Jeffrey R. Johnson & Neil L. Kelleher

Authors
  1. Fanyu Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Benjamin J. Cargile
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leah M. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew J. Forbes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey R. Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Neil L. Kelleher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Neil L. Kelleher.

Supplementary information

Supplementary Tables 1–6 (PDF 15 kb)

41587_2001_BFnbt1001952_MOESM2_ESM.gif

Supplementary Figure 1. Irradiation of the two components of Figure 2 with infrared photons for 125 ms (A), 225 ms (B), and 325 ms (C); 9.4 T data, each 25 scans. (GIF 62 kb)

Supplementary Experimental Protocol (PDF 33 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Meng, F., Cargile, B., Miller, L. et al. Informatics and multiplexing of intact protein identification in bacteria and the archaea. Nat Biotechnol 19, 952–957 (2001). https://doi.org/10.1038/nbt1001-952

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1001-952

This article is cited by

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