Two-dimensional gel electrophoresis in platelet proteomics research.
Proteomics technology allows a comprehensive and efficient analysis of the proteome and has become an indispensable tool in biomedical research. Since the late 80s, advances on mass spectrometry (MS) instrumentation and techniques have revolutionized the way proteins can be analyzed. Such analysis would only be possible with a proper sample preparation and separation ahead of the MS step. Different gel and nongel-based methods are available for protein separation. This chapter will focus on the use of two-dimensional gel electrophoresis (2-DE) in proteomics and its application to platelet research. 2-DE separates proteins according to their isoelectric point (pI) and size (molecular weight) and allows the detection of thousands of proteins at a time. Platelets are enucleated cells that play a critical function in the control of bleeding and wound healing. As platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. This chapter presents a protocol for an efficient sample preparation, protein separation by 2-DE, and protein digestion ahead of the MS analysis. The experimental approach, already successfully applied to the study of the platelet proteome, includes recommendations for an efficient platelet preparation for proteomics studies.
García
Angel
A
Department of Biochemistry, Rede de Infraestructuras de Apoio á Investigación e ó Desenvolvemento Tecnolóxico (RIAIDT), Universidade de Santiago de Compostela, Spain.
D001792:Blood Platelets / Q000737:chemistry* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000379:methods*