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

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Applications of MALDI-TOF Spectroscopy

Part of the book series: Topics in Current Chemistry ((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.

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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

References

  1. Karas M, Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60:2299–2301

    Article  CAS  Google Scholar 

  2. Hillenkamp F, Karas M (1990) Mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/ionization. Methods Enzymol 193:280–295

    Article  CAS  Google Scholar 

  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 62:793–796

    Article  CAS  Google Scholar 

  4. Cohen LH, Gusev AI (2002) Small molecule analysis by MALDI mass spectrometry. Anal Bioanal Chem 373:571–586

    Article  CAS  Google Scholar 

  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 71:3238–3247

    Article  CAS  Google Scholar 

  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 11:513–518

    Article  CAS  Google Scholar 

  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 301:22–28

    Article  CAS  Google Scholar 

  8. Aebersold R, Goodlett DR (2001) Mass spectrometry in proteomics. Chem Rev 101:269–295

    Article  CAS  Google Scholar 

  9. Jungblut P, Thiede B (1997) Protein identification from 2-DE gels by MALDI mass spectrometry. Mass Spectrom Rev 16:145–162

    Article  CAS  Google Scholar 

  10. Krutchinsky AN, Kalkum M, Chait BT (2001) Automatic identification of proteins with a MALDI-quadrupole ion trap mass spectrometer. Anal Chem 73:5066–5077

    Article  CAS  Google Scholar 

  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 389:1075–1085

    Article  CAS  Google Scholar 

  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):1425–1433

    Article  CAS  Google Scholar 

  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 8:107

    Article  CAS  Google Scholar 

  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 48:178–182

    Article  CAS  Google Scholar 

  15. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567

    Article  CAS  Google Scholar 

  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 9:255–262

    Article  CAS  Google Scholar 

  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 9:28–32

    Article  CAS  Google Scholar 

  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 22:881–886

    Article  CAS  Google Scholar 

  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 390:1765–1773

    Article  CAS  Google Scholar 

  20. Ladner CL, Yang J, Turner RJ, Edwards RA (2004) Visible fluorescent detection of proteins in polyacrylamide gels without staining. Anal Biochem 326:13–20

    Article  CAS  Google Scholar 

  21. Sluszny C, Yeung ES (2004) One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection. Anal Chem 76:1359–1365

    Article  CAS  Google Scholar 

  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 18:331–335

    Google Scholar 

  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 75:157–159

    Article  CAS  Google Scholar 

  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 305:217–227

    Google Scholar 

  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 23:249–261

    Article  CAS  Google Scholar 

  26. Koohmaraie M (1996) Biochemical factors regulating the toughening and tenderization processes of meat. Meat Sci 43:193–201

    Article  Google Scholar 

  27. Huang J, Forsberg NE (1998) Role of calpain in skeletal-muscle protein degradation. Proc Natl Acad Sci USA 95:12100–12105

    Article  CAS  Google Scholar 

  28. Doumit ME, Koohmaraie M (1999) Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle. J Anim Sci 77:1467–1473

    CAS  Google Scholar 

  29. Lametsch R, Roepstorff P, Bendixen E (2002) Identification of protein degradation during post-mortem storage of pig meat. J Agric Food Chem 50:5508–5512

    Article  CAS  Google Scholar 

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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).

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Authors and Affiliations

  1. Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, 78457, Konstanz, Germany

    Iuliana Susnea, Bogdan Bernevic, Li Ma & Michael Przybylski

  2. Institute of Animal Breeding and Genetics, University of Göttingen, Göttingen, Germany

    Michael Wicke

  3. Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, People’s Republic of China

    Li Ma & Shuying Liu

  4. Department of Animal Physiology and Veterinary Medicine, University of Bonn, Bonn, Germany

    Karl Schellander

Authors
  1. Iuliana Susnea
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  2. Bogdan Bernevic
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  3. Michael Wicke
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  4. Li Ma
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  5. Shuying Liu
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  6. Karl Schellander
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  7. Michael Przybylski
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Corresponding author

Correspondence to Michael Przybylski .

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Editors and Affiliations

  1. , Department of Chemistry, Hong Kong Baptist University, Ho Sin Hang Campus, Hong Kong, 000000000, China, People's Republic

    Zongwei Cai

  2. , Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 000000000, China, People's Republic

    Shuying Liu

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© 2012 Springer-Verlag Berlin Heidelberg

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Susnea, I. et al. (2012). Application of MALDI-TOF-Mass Spectrometry to Proteome Analysis Using Stain-Free Gel Electrophoresis. In: Cai, Z., Liu, S. (eds) Applications of MALDI-TOF Spectroscopy. Topics in Current Chemistry, vol 331. Springer, Berlin, Heidelberg. https://doi.org/10.1007/128_2012_321

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