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

Abstract

Publisher Summary 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 this approach can be fractionated by rapid (∼ 10 min) reversed-phase chromatography using conventional silica-based supports and standard liquid chromatographs. Although, these supports are commercially available, they have not gained general acceptance due to serious drawbacks, such as low mass loading (capacity) and high back pressure. The chapter explains that in conjunction with mass spectrometric peptide-mass fingerprinting technologies, this approach may allow a rapid expansion of 2D gel protein databases.