Internal Peptide Sequence of Proteins Digested In-Gel after One- or Two- Dimensional Gel Electrophoresis

This chapter focuses on an internal peptide sequence of proteins digested in-gel after one- or two- dimensional gel electrophoresis. The two most employed electrophoretic techniques, SDS-PAGE and 2D-GE, when used in micropreparative scale, are powerful tools for the isolation of microgram quantities of proteins. A modified CBB staining protocol was developed followed by a simple in-gel digestion technique, which allowed obtaining internal peptide sequences from 1–2 μg of protein separated by SDS-PAGE and 2D-GE. The method is rapid and allows the use of SDS-sensitive and SDS-resistant proteases. The in-gel digestion overcomes the electrotransfer or electroelution step and the need for the elution of peptides from PVDF membranes or glass-fiber filters, reducing considerably the material losses and the risk of chemical amino acid damage. The chapter describes the identification, through internal sequence analysis, of E. coli proteins separated on SDS-PAGE, and of rat heart mitochondrial membrane proteins separated on giant 2D gels.