Advertisement

Detection and Identification of Low-Abundant Proteins Using HPE Gels, Fluorescent Stains, and MALDI-ToF-ToF-MS

  • Martin Moche
  • Dirk Albrecht
  • Reiner Westermeier
  • Knut Büttner
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1841)

Abstract

Two-dimensional electrophoresis as a complementary approach to gel-free proteomic methods possesses the ability to separate physiologically important isoforms of proteins in an unbiased manner. Frequently, those isoforms are low-abundant regulators, and therefore, detection and identification of low-abundant proteins is highly necessary to exploit this advantage. We describe an experimental sequence of classical operations to process gels but optimized them, in order to identify each detectable protein spot on gel.

Key words

High performance electrophoresis Low-abundant proteins Fluorescent stains Scanning conditions Tryptic digestion MALDI-ToF-MS 

References

  1. 1.
    Hecker M, Antelmann H, Büttner K, Bernhardt J (2008) Gel-based proteomics of Gram-positive bacteria: a powerful tool to address physiological questions. Proteomics 8(23-24):4958–4975CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250(10):4007–4021PubMedPubMedCentralGoogle Scholar
  3. 3.
    Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26(3):231–243PubMedGoogle Scholar
  4. 4.
    Muntel J, Fromion V, Goelzer A, Maabeta S, Mader U, Büttner K, Hecker M, Becher D (2014) Comprehensive absolute quantification of the cytosolic proteome of Bacillus subtilis by data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MS(E)). Mol Cell Proteomics 13(4):1008–1019CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Markert S, Arndt C, Felbeck H, Becher D, Sievert SM, Hugler M, Albrecht D, Robidart J, Bench S, Feldman RA, Hecker M, Schweder T (2007) Physiological proteomics of the uncultured endosymbiont of Riftia pachyptila. Science 315(5809):247–250CrossRefPubMedGoogle Scholar
  6. 6.
    Moche M, Albrecht D, Maass S, Hecker M, Westermeier R, Büttner K (2013) The new horizon in 2D electrophoresis: new technology to increase resolution and sensitivity. Electrophoresis 34(11):1510–1518CrossRefPubMedGoogle Scholar
  7. 7.
    Bjellqvist B, Ek K, Righetti PG, Gianazza E, Gorg A, Westermeier R, Postel W (1982) Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J Biochem Biophys Methods 6(4):317–339CrossRefPubMedGoogle Scholar
  8. 8.
    Hessling B, Büttner K, Hecker M, Becher D (2013) Global relative quantification with liquid chromatography-matrix-assisted laser desorption ionization time-of-flight (LC-MALDI-TOF)—cross-validation with LTQ-Orbitrap proves reliability and reveals complementary ionization preferences. Mol Cell Proteomics 12(10):2911–2920CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Griebel A, Obermaier C, Westermeier R, Moche M, Büttner K (2013) Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE lightning red. Arch Physiol Biochem 119(3):94–99CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Martin Moche
    • 1
  • Dirk Albrecht
    • 2
  • Reiner Westermeier
    • 3
  • Knut Büttner
    • 2
  1. 1.Waters GmbHEschbornGermany
  2. 2.Department of Microbial Physiology and Molecular Biology, Institute for MicrobiologyUniversity GreifswaldGreifswaldGermany
  3. 3.FreisingGermany

Personalised recommendations