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Sample Preparation for 2DE Using Samples of Animal Origin

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Proteomics in Domestic Animals: from Farm to Systems Biology

Abstract

Protein separation by two-dimensional gel electrophoresis (2DE) is a well-established technique for proteome analysis. Despite the enormous potential of this methodology for resolving proteins in complex samples, the fact is that its use is limited and often avoided due to inadequate sample preparation. It is understood as being especially critical for 2DE-based proteomics research that samples need to be of good quality, and this goal could be accomplished by optimizing or adapting the sample preparation protocols to the specificities (i.e., chemical composition) of biological samples. Sample preparation protocols should also comply with the objectives of the research. For selection of the adequate protocol and for its optimization, it is therefore important first to know the distinct steps that constitute a protocol and the main objectives underlying each step. Secondly, it is important to understand the potential limitations of the biological samples hence to perform the necessary adjustments/alterations to the protocol. This chapter aims to review the major steps of sample preparation protocols for 2DE with particular focus on optimization, taking into account specificities of the biological samples. The main technical aspects concerning sample tissue disruption and cell lysis, protein extraction, fractionation, enrichment, and solubilization prior to 2DE will be presented and discussed. In this chapter, the most common biological samples employed in proteomics and 2DE research and corresponding sample preparation methods will be reviewed. This follows a brief discussion on 2DE and MALDI-TOF approaches. A selection of methods for 2DE sample preparation is included based on the authors’ experience.

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Abbreviations

2DE:

Two-dimensional gel electrophoresis

CHAPS:

3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate

CPLL:

Combinatorial peptide ligand libraries

DDM:

n-Dodecyl β-d-maltoside

DTT:

Dithiothreitol

HPLC:

High-performance liquid chromatography

IAA:

Iodoacetamide

IEF:

Isoelectric focusing

LC-MS:

Liquid chromatography coupled with mass spectrometry

MALDI-TOF:

Matrix-assisted laser desorption/ionization—time of flight

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Acknowledgments

A. Campos’ work is supported by postdoctoral grant (SFRH/BPD/103683/2014) from FCT. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work was financed by FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the COMPETE 2020, Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and Portuguese funds through FCT (Foundation for Science and Technology/Ministério da Ciência, Tecnologia e Inovação) in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), “PEst-C/SAU/LA0003/2013” and “UID/multi/04423/2013”.

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Authors and Affiliations

  1. i3s-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

    Hugo Osório

  2. IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal

    Hugo Osório

  3. Faculty of Medicine, Department of Pathology and Oncology, University of Porto, Porto, Portugal

    Hugo Osório

  4. Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal

    André M. de Almeida

  5. CIIMAR – Interdisciplinary Centre of Marine and Environmental Sciences, University of Porto, Matosinhos, Portugal

    Alexandre Campos

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  1. Hugo Osório
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  2. André M. de Almeida
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  3. Alexandre Campos
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Correspondence to Alexandre Campos .

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Editors and Affiliations

  1. Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, Lisbon, Portugal

    Andre Martinho de Almeida

  2. ERA Chair Laboratory, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia

    David Eckersall

  3. Institute of Medical Biochemistry, University of Veterinary Medicine, Vienna, Austria

    Ingrid Miller

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Osório, H., de Almeida, A.M., Campos, A. (2018). Sample Preparation for 2DE Using Samples of Animal Origin. In: de Almeida, A., Eckersall, D., Miller, I. (eds) Proteomics in Domestic Animals: from Farm to Systems Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-69682-9_4

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