Analytical and Bioanalytical Chemistry

, Volume 386, Issue 1, pp 92–103 | Cite as

Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry

  • Olaf Jahn
  • Dörte Hesse
  • Marina Reinelt
  • Hartmut D. Kratzin
Special Issue Paper


The combination of gel-based two-dimensional protein separations with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the workhorse for the large-scale analyses of proteomes. Such high-throughput proteomic approaches require automation of all post-separation steps and the in-gel digest of proteins especially is often the bottleneck in the protein identification workflow. With the objective of reaching the same high performance of manual low-throughput in-gel digest procedures, we have developed a novel stack-type digestion device and implemented it into a commercially available robotic liquid handling system. This modified system is capable of performing in-gel digest, extraction of proteolytic peptides, and subsequent sample preparation for MALDI-MS without any manual intervention, but with a performance at least identical to manual procedures as indicated on the basis of the sequence coverage obtained by peptide mass fingerprinting. For further refinement of the automated protein identification workflow, we have also developed a motor-operated matrix application device to reproducibly obtain homogenous matrix preparation of high quality. This matrix preparation was found to be suitable for the automated acquisition of both peptide mass fingerprint and fragment ion spectra from the same sample spot, a prerequisite for high confidence protein identifications on the basis of peptide mass and sequence information. Due to the implementation of the stack-type digestion device and the motor-operated matrix application device, the entire platform works in a reliable, cost-effective, and sensitive manner, yielding high confidence protein identifications even for samples in the concentration range of as low as 100 fmol protein per gel plug.


Proteomics Automation In-gel digest Sample preparation Matrix MALDI mass spectrometry 



This study was supported by the Max Planck Society and the DFG Research Center for Molecular Physiology of the Brain (CMPB). Markus Krohn and all other members of the mechanics workshop of the MPI of Experimental Medicine are gratefully acknowledged for building the hardware components and thereby realizing our ideas. We also thank Gabriele Paetzold, Thomas Liepold, and Lars Piepkorn for excellent assistance, as well as Kalina Dimova, Bernhard Schmidt, and the members of the Goettingen Proteomics Forum ( for stimulating discussions.


  1. 1.
    Gorg A, Weiss W, Dunn MJ (2004) Proteomics 4:3665–3685CrossRefGoogle Scholar
  2. 2.
    Van den Bergh G, Arckens L (2005) Expert Rev Proteomics 2:243–252CrossRefGoogle Scholar
  3. 3.
    Taylor CM, Pfeiffer SE (2003) Proteomics 3:1303–1312CrossRefGoogle Scholar
  4. 4.
    Patton WF (2002) J Chromatogr B Analyt Technol Biomed Life Sci 771:3–31CrossRefGoogle Scholar
  5. 5.
    Stasyk T, Huber LA (2004) Proteomics 4:3704–3716CrossRefGoogle Scholar
  6. 6.
    Hartinger J, Stenius K, Hogemann D, Jahn R (1996) Anal Biochem 240:126–133CrossRefGoogle Scholar
  7. 7.
    Zahedi RP, Meisinger C, Sickmann A (2005) Proteomics 5:3581–3588CrossRefGoogle Scholar
  8. 8.
    Navarre C, Degand H, Bennett KL, Crawford JS, Mortz E, Boutry M (2002) Proteomics 2:1706–1714CrossRefGoogle Scholar
  9. 9.
    Rais I, Karas M, Schagger H (2004) Proteomics 4:2567–2571CrossRefGoogle Scholar
  10. 10.
    Yergey AL, Coorssen JR, Backlund PS Jr, Blank PS, Humphrey GA, Zimmerberg J, Campbell JM, Vestal ML (2002) J Am Soc Mass Spectrom 13:784–791CrossRefGoogle Scholar
  11. 11.
    Bienvenut WV, Deon C, Pasquarello C, Campbell JM, Sanchez JC, Vestal ML, Hochstrasser DF (2002) Proteomics 2:868–876CrossRefGoogle Scholar
  12. 12.
    Suckau D, Resemann A, Schuerenberg M, Hufnagel P, Franzen J, Holle A (2003) Anal Bioanal Chem 376:952–965CrossRefGoogle Scholar
  13. 13.
    Vestal ML, Campbell JM (2005) Methods Enzymol 402:79–108Google Scholar
  14. 14.
    Pappin DJ, Hojrup P, Bleasby AJ (1993) Curr Biol 3:327–332CrossRefGoogle Scholar
  15. 15.
    Thiede B, Hohenwarter W, Krah A, Mattow J, Schmid M, Schmidt F, Jungblut PR (2005) Methods 35:237–247CrossRefGoogle Scholar
  16. 16.
    Nordhoff E, Egelhofer V, Giavalisco P, Eickhoff H, Horn M, Przewieslik T, Theiss D, Schneider U, Lehrach H, Gobom J (2001) Electrophoresis 22:2844–2855CrossRefGoogle Scholar
  17. 17.
    Cramer R, Gobom J, Nordhoff E (2005) Expert Rev Proteomics 2:407–420CrossRefGoogle Scholar
  18. 18.
    Quadroni M, James P (1999) Electrophoresis 20:664–677CrossRefGoogle Scholar
  19. 19.
    Houthaeve T, Gausepohl H, Mann M, Ashman K (1995) FEBS Lett 376:91–94CrossRefGoogle Scholar
  20. 20.
    Houthaeve T, Gausepohl H, Ashman K, Nillson T, Mann M (1997) J Protein Chem 16:343–348CrossRefGoogle Scholar
  21. 21.
    Traini M, Gooley AA, Ou K, Wilkins MR, Tonella L, Sanchez JC, Hochstrasser DF, Williams KL (1998) Electrophoresis 19:1941–1949CrossRefGoogle Scholar
  22. 22.
    Mooney BP, Krishnan HB, Thelen JJ (2004) Phytochemistry 65:1733–1744CrossRefGoogle Scholar
  23. 23.
    Gobom J, Schuerenberg M, Mueller M, Theiss D, Lehrach H, Nordhoff E (2001) Anal Chem 73:434–438CrossRefGoogle Scholar
  24. 24.
    Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Electrophoresis 9:255–262CrossRefGoogle Scholar
  25. 25.
    Luo S, Wehr NB, Levine RL (2006) Anal Biochem 350:233–238CrossRefGoogle Scholar
  26. 26.
    Nordhoff E, Schurenberg M, Thiele G, Lubbert C, Kloeppel KD, Theiss D, Lehrach H, Gobom J (2003) Int J Mass Spectrom 226:163–180CrossRefGoogle Scholar
  27. 27.
    Mirgorodskaya E, Braeuer C, Fucini P, Lehrach H, Gobom J (2005) Proteomics 5:399–408CrossRefGoogle Scholar
  28. 28.
    Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Electrophoresis 20:3551–3567CrossRefGoogle Scholar
  29. 29.
    Katayama H, Nagasu T, Oda Y (2001) Rapid Commun Mass Spectrom 15:1416–1421CrossRefGoogle Scholar
  30. 30.
    van Montfort BA, Canas B, Duurkens R, Godovac-Zimmermann J, Robillard GT (2002) J Mass Spectrom 37:322–330CrossRefGoogle Scholar
  31. 31.
    Cohen SL, Chait BT (1996) Anal Chem 68:31–37CrossRefGoogle Scholar
  32. 32.
    Russell WK, Park ZY, Russell DH (2001) Anal Chem 73:2682–2685CrossRefGoogle Scholar
  33. 33.
    Castellanos-Serra L, Ramos Y, Huerta V (2005) Proteomics 5:2729–2738CrossRefGoogle Scholar
  34. 34.
    Lopez MF, Kristal BS, Chernokalskaya E, Lazarev A, Shestopalov AI, Bogdanova A, Robinson M (2000) Electrophoresis 21:3427–3440CrossRefGoogle Scholar
  35. 35.
    Finehout EJ, Lee KH (2003) Electrophoresis 24:3508–3516CrossRefGoogle Scholar
  36. 36.
    Ma C, Haslbeck M, Babujee L, Jahn O, Reumann S (2006) Plant Physiol 141:47–60CrossRefGoogle Scholar
  37. 37.
    Kyte J, Doolittle RF (1982) J Mol Biol 157:105–132CrossRefGoogle Scholar
  38. 38.
    Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) Nucleic Acids Res 31:3784–3788CrossRefGoogle Scholar
  39. 39.
    Yu W, Vath JE, Huberty MC, Martin SA (1993) Anal Chem 65:3015–3023CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Olaf Jahn
    • 1
    • 2
    • 4
  • Dörte Hesse
    • 1
    • 3
  • Marina Reinelt
    • 1
    • 2
    • 4
  • Hartmut D. Kratzin
    • 1
  1. 1.Proteomics GroupMax-Planck-Institute of Experimental MedicineGöttingenGermany
  2. 2.Department of Molecular NeurobiologyMax-Planck-Institute of Experimental MedicineGöttingenGermany
  3. 3.Department of NeurogeneticsMax-Planck-Institute of Experimental MedicineGöttingenGermany
  4. 4.DFG Research Center for Molecular Physiology of the Brain (CMPB)GöttingenGermany

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