Rapid separation of proteins and peptides using conventional silica-based supports: Identification of 2-D gel proteins following in-gel proteolysis

Abstract

Rapid chromatographic separation of proteins and peptides at high flow velocities on silica-based supports is not a new concept; however, these experiments are performed on specifically designed supports. This chapter describes a protocol for fast chromatographic analysis of proteins and peptides using conventional wide pore supports and standard liquid chromatographs, and presents a modified procedure for performing in-gel proteolytic digestion of 2-D gel protein spots for the purpose of identifying proteins by peptide-mass fingerprinting along with micro-sequencing. Peptides can be recovered from acrylamide gel pieces, following in-gel proteolysis, by TFA extraction. Peptides obtained by t..