Enzymatic digestion of PVDF-bound proteins in the presence of glucopyranoside detergents: Applicability to mass spectrometry

Abstract

Publisher Summary Amino terminal protein sequence analysis is a vital tool in understanding the functions of proteins, and enzymatic digestion of PVDF-bound proteins in the presence of hydrogenated Triton X-100 (RTX-100) followed by microbore HPLC purification of the peptide fragments is an efficient and sensitive procedure to obtain internal protein sequence data. This chapter discusses a study in which RTX-100 is substituted by heptyl, octyl, nonyl, and decyl glucopyranosides, and presents a discussion on peptide recoveries, amino terminal sequence analysis, and MALDI-TOF mass spectrometry of the recovered peptides and the detergent containing peptide mixtures. The results show that octylglucopyranoside can be substituted for RTX-100 with only a slight decrease in peptide recoveries, and decylglucopyranoside may be used in place of RTX-100, when acetonitrile is added to the digestion buffer, however, it is not useful for endoproteinase Glu-C. Heptyl and nonyl glucopyranosides are not suitable alternatives as there is no peptides recovered with the former and micelle formation occurred with the latter. Thus, following HPLC purification of transferrin digestions, no difference in sequencing initial yields of the recovered peptides between octylglucopyranoside, decylglucopyranoside, and RTX-100 is observed.