Advertisement

Application of MALDI-TOF-Mass Spectrometry to Proteome Analysis Using Stain-Free Gel Electrophoresis

  • Iuliana Susnea
  • Bogdan Bernevic
  • Michael Wicke
  • Li Ma
  • Shuying Liu
  • Karl Schellander
  • Michael Przybylski
Chapter
Part of the Topics in Current Chemistry book series (TOPCURRCHEM, volume 331)

Abstract

The combination of MALDI-TOF-mass spectrometry with gel electrophoretic separation using protein visualization by staining procedures involving such as Coomassie Brilliant Blue has been established as a widely used approach in proteomics. Although this approach has been shown to present high detection sensitivity, drawbacks and limitations frequently arise from the significant background in the mass spectrometric analysis. In this chapter we describe an approach for the application of MALDI-MS to the mass spectrometric identification of proteins from one-dimensional (1D) and two-dimensional (2D) gel electrophoretic separation, using stain-free detection and visualization based on native protein fluorescence. Using the native fluorescence of aromatic protein amino acids with UV transmission at 343 nm as a fast gel imaging system, unstained protein spots are localized and, upon excision from gels, can be proteolytically digested and analyzed by MALDI-MS. Following the initial development and testing with standard proteins, applications of the stain-free gel electrophoretic detection approach to mass spectrometric identification of biological proteins from 2D-gel separations clearly show the feasibility and efficiency of this combination, as illustrated by a proteomics study of porcine skeleton muscle proteins. Major advantages of the stain-free gel detection approach with MALDI-MS analysis are (1) rapid analysis of proteins from 1D- and 2D-gel separation without destaining required prior to proteolytic digestion, (2) the low detection limits of proteins attained, and (3) low background in the MALDI-MS analysis.

Keywords

Gel electrophoresis MALDI-TOF-mass spectrometry Native fluorescence Protein identification Skeleton muscle proteomics 

Abbreviations

1D

One-dimensional gel electrophoresis

2D

Two-dimensional gel electrophoresis

MALDI-TOF

Matrix assisted laser desorption/ionization–time-of-flight

MS

Mass spectrometry

PMF

Peptide mass fingerprinting

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Notes

Acknowledgments

We thank Martin Schütte and Bernd Müller-Zülow, LaVision-BioTec for technical support regarding the gel bioanalyzer. This work has been partially supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (PR-175-14/1), and the University of Konstanz (Proteostasis Research Center).

References

  1. 1.
    Karas M, Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60:2299–2301CrossRefGoogle Scholar
  2. 2.
    Hillenkamp F, Karas M (1990) Mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/ionization. Methods Enzymol 193:280–295CrossRefGoogle Scholar
  3. 3.
    Spengler B, Cotter RJ (1990) Ultraviolet laser desorption/ionization mass spectrometry of proteins above 100,000 daltons by pulsed ion extraction time-of-flight analysis. Anal Chem 62:793–796CrossRefGoogle Scholar
  4. 4.
    Cohen LH, Gusev AI (2002) Small molecule analysis by MALDI mass spectrometry. Anal Bioanal Chem 373:571–586CrossRefGoogle Scholar
  5. 5.
    Schleuder D, Hillenkamp F, Strupat K (1999) IR-MALDI-mass analysis of electroblotted proteins directly from the membrane: comparison of different membranes, application to on-membrane digestion, and protein identification by database searching. Anal Chem 71:3238–3247CrossRefGoogle Scholar
  6. 6.
    Petre BA, Youhnovski N, Lukkari J, Weber R, Przybylski M (2005) Structural characterisation of tyrosine-nitrated peptides by ultraviolet and infrared matrix-assisted laser desorption/ionisation Fourier transform ion cyclotron resonance mass spectrometry. Eur J Mass Spectrom 11:513–518CrossRefGoogle Scholar
  7. 7.
    Susnea I, Bernevic B, Svobodova E, Simeonova DD, Wicke M, Werner C, Schink B, Przybylski M (2011) Mass spectrometric protein identification from two-dimensional gel separation with stain-free detection and visualization using native fluorescence. Int J Mass Spectrom 301:22–28CrossRefGoogle Scholar
  8. 8.
    Aebersold R, Goodlett DR (2001) Mass spectrometry in proteomics. Chem Rev 101:269–295CrossRefGoogle Scholar
  9. 9.
    Jungblut P, Thiede B (1997) Protein identification from 2-DE gels by MALDI mass spectrometry. Mass Spectrom Rev 16:145–162CrossRefGoogle Scholar
  10. 10.
    Krutchinsky AN, Kalkum M, Chait BT (2001) Automatic identification of proteins with a MALDI-quadrupole ion trap mass spectrometer. Anal Chem 73:5066–5077CrossRefGoogle Scholar
  11. 11.
    Bai Y, Galetskiy D, Damoc E, Ripper J, Woischnik M, Griese M, Liu Z, Liu S, Przybylski M (2007) Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry. Anal Bioanal Chem 389:1075–1085CrossRefGoogle Scholar
  12. 12.
    Damoc E, Youhnovski N, Crettaz D, Tissot JD, Przybylski M (2003) High resolution proteome analysis of cryoglobulins using Fourier transform-ion cyclotron resonance mass spectrometry. Proteomics 3(8):1425–1433CrossRefGoogle Scholar
  13. 13.
    Sun JF, Shi ZX, Guo HC, Li S, Tu CC (2011) Proteomic analysis of swine serum following highly virulent classical swine fever virus infection. Virol J 8:107CrossRefGoogle Scholar
  14. 14.
    Takagi T, Naito Y, Okada H, Okayama T, Mizushima K, Yamada S, Fukumoto K, Inoue K, Takaoka M, Oya-Ito T, Uchiyama K, Ishikawa T, Handa O, Kokura S, Yagi N, Ichikawa H, Kato Y, Osawa T, Yoshikawa T (2011) Identification of dihalogenated proteins in rat intestinal mucosa injured by indomethacin. J Clin Biochem Nutr 48:178–182CrossRefGoogle Scholar
  15. 15.
    Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567CrossRefGoogle Scholar
  16. 16.
    Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262CrossRefGoogle Scholar
  17. 17.
    Heukeshoven J, Dernick R (1988) Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis 9:28–32CrossRefGoogle Scholar
  18. 18.
    Nock CM, Ball MS, White IR, Skehel JM, Bill L, Karuso P (2008) Mass spectrometric compatibility of Deep Purple and SYPRO Ruby total protein stains for high-throughput proteomics using large-format two-dimensional gel electrophoresis. Rapid Commun Mass Spectrom 22:881–886CrossRefGoogle Scholar
  19. 19.
    Lin JF, Chen QX, Tian HY, Gao X, Yu ML, Xu GJ, Zhao FK (2008) Stain efficiency and MALDI-TOF MS compatibility of seven visible staining procedures. Anal Bioanal Chem 390:1765–1773CrossRefGoogle Scholar
  20. 20.
    Ladner CL, Yang J, Turner RJ, Edwards RA (2004) Visible fluorescent detection of proteins in polyacrylamide gels without staining. Anal Biochem 326:13–20CrossRefGoogle Scholar
  21. 21.
    Sluszny C, Yeung ES (2004) One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection. Anal Chem 76:1359–1365CrossRefGoogle Scholar
  22. 22.
    Zhao Z, Aliwarga Y, Willcox MD (2007) Intrinsic protein fluorescence interferes with detection of tear glycoproteins in SDS-polyacrylamide gels using extrinsic fluorescent dyes. J Biomol Tech 18:331–335Google Scholar
  23. 23.
    Roegener J, Lutter P, Reinhardt R, Bluggel M, Meyer HE, Anselmetti D (2003) Ultrasensitive detection of unstained proteins in acrylamide gels by native UV fluorescence. Anal Chem 75:157–159CrossRefGoogle Scholar
  24. 24.
    Bernevic B, Petre BA, Galetskiy D, Werner C, Wicke M, Schellander K, Przybylski M (2010) Degradation and oxidation postmortem of myofibrillar proteins in porcine skeleton muscle revealed by high resolution mass spectrometric proteome analysis. Int J Mass Spectrom 305:217–227Google Scholar
  25. 25.
    Mortz E, Vorm O, Mann M, Roepstorff P (1994) Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search. Biol Mass Spectrom 23:249–261CrossRefGoogle Scholar
  26. 26.
    Koohmaraie M (1996) Biochemical factors regulating the toughening and tenderization processes of meat. Meat Sci 43:193–201CrossRefGoogle Scholar
  27. 27.
    Huang J, Forsberg NE (1998) Role of calpain in skeletal-muscle protein degradation. Proc Natl Acad Sci USA 95:12100–12105CrossRefGoogle Scholar
  28. 28.
    Doumit ME, Koohmaraie M (1999) Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle. J Anim Sci 77:1467–1473Google Scholar
  29. 29.
    Lametsch R, Roepstorff P, Bendixen E (2002) Identification of protein degradation during post-mortem storage of pig meat. J Agric Food Chem 50:5508–5512CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Iuliana Susnea
    • 1
  • Bogdan Bernevic
    • 1
  • Michael Wicke
    • 2
  • Li Ma
    • 1
    • 3
  • Shuying Liu
    • 3
  • Karl Schellander
    • 4
  • Michael Przybylski
    • 1
  1. 1.Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of ChemistryUniversity of KonstanzKonstanzGermany
  2. 2.Institute of Animal Breeding and GeneticsUniversity of GöttingenGöttingenGermany
  3. 3.Changchun Institute of Applied ChemistryChinese Academy of SciencesChangchunPeople’s Republic of China
  4. 4.Department of Animal Physiology and Veterinary MedicineUniversity of BonnBonnGermany

Personalised recommendations