Abstract
Two-dimensional electrophoresis (2-DE) is a classical separation technique for complex protein mixtures based on a first-separation step by isoelectric point (pI) followed by SDS-PAGE. Due to its high resolving power (including protein isoforms) and the capability to display post-translational modifications, 2-DE has been the technique of choice in many studies focused in characterizing allergens. In this chapter, we describe a standard protocol which enables efficient protein separation and subsequent protein identification by tandem mass spectrometry (MALDI-MS/MS).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bruno B (2012) Two-dimensional gel electrophoresis (2-DE), gel electrophoresis—principles and basics. In: Sameh M (ed) InTech. doi: 10.5772/38312
Monaci L, Visconti A (2009) Mass spectrometry-based proteomics methods for analysis of food allergens. Trends Analyt Chem 28(5):581–591
Castro L, Crespo JF, Rodríguez J et al (2015) Immunoproteomic tools are used to identify masked allergens: Ole e 12, an allergenic isoflavone reductase from olive (Olea europaea) pollen. Biochim Biophys Acta 1854(12):1871–1880
Abou Chakra OR, Sutra JP, Demey Thomas E et al (2012) Proteomic analysis of major and minor allergens from isolated pollen cytoplasmic granules. J Proteome Res 11(2):1208–1216
Perez-Gordo M, Cuesta-Herranz J, Maroto AS et al (2011) Identification of sole parvalbumin as a major allergen: study of cross-reactivity between parvalbumins in a Spanish fish-allergic population. Clin Exp Allergy 41(5):750–758
Unlü M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18(11):2071–2077
Ausubel FM, Brent R, Kingston RE et al (2005) Current Protocols in Molecular Biology, vol 10. John Wiley & Sons, New York, NY, pp 97–106
Gorg A, Weiss W, Dunn MJ (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4(12):3665–3685
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Barrio E, Antón L, Marques G, Sánchez A, Vivanco F (1991) Formation of covalently linked C3-C3 dimers on IgG immune aggregates. Eur J Immunol 21(2):343–349
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856–2860
Acknowledgments
This work was supported by grants from the Instituto de Salud Carlos III (FIS PI070537, IF08/3667-1, PI11-02239, PI14/01917, PI11/01401, PI11/02432, PI13/01873, PI13/01746, PI13/01581, PI14/01650, PI14/01841) by PT13/0001/0013, IDCSalud (3371/002) and Redes Temáticas de Investigación Cooperativa (FONDOS FEDER, RD06/0014/1015, RD12/0042/0071).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
de la Cuesta, F., Alvarez-Llamas, G., Barderas, M.G. (2017). Two-Dimensional Electrophoresis and Identification by Mass Spectrometry. In: Lin, J., Alcocer, M. (eds) Food Allergens. Methods in Molecular Biology, vol 1592. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6925-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6925-8_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6923-4
Online ISBN: 978-1-4939-6925-8
eBook Packages: Springer Protocols