Proteomics and laser microdissection.
Two-dimensional gel electrophoresis (2-DE) combined with protein identification by mass spectrometry (MS) is currently the method of choice in the majority of proteomic projects. Novel gel-free technologies have been developed but 2-DE remains the technique of choice for quantitative expression profiling of large sets of complex protein mixtures such as whole cell/tissue lysates. Solubilized proteins are separated in the first dimension according to their charge properties (isoelectric point, pI) by isoelectric focusing (IEF) under denaturing conditions, followed by their separation in the second dimension by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to their relative molecular mass (Mr). 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely) and can detect less than 1 ng of protein per spot. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or posttranslational modifications. In this chapter we describe the various steps in the 2-DE proteomics workflow, namely sample preparation, solubilization, 2-D gel electrophoresis, protein detection and visualization, and protein identification by mass spectrometry. The use of 2-DE in conjunction with laser microdissection microscopy is presented and discussed.
McGregor
Emma
E
Department of Vascular Surgery, Imperial College School of Medicine, Charing Cross Hospital, London, England.
De Souza
Ayesha
A
D000818:Animals; D006801:Humans; D007834:Lasers; D042282:Microdissection; D040901:Proteomics / Q000379:methods*