Reversed-phase liquid chromatography of peptides for direct micro-sequencing

Tryptic and cyanogen bromide peptides derived from yeast aspartyl-tRNA synthetase and from Escherichia coli ribosomal proteins were separated by reversed-phase liquid chromatography, employing volatile buffers of low ionic strength. The conditions used allow the performance of micro-sequencing without desalting or extensive lyophilization, and can therefore be applied to peptide mixtures containing hydrophobic fragments which tend to precipitate. To prevent losses of peptides, direct ultra-violet detection of the peptides was preferred, to detection by post-column derivatization with an additional stream splitting device. Preparative separations were performed with 5-10 nmol of peptide mixture; analytical runs were made with 5-10 micrograms of protein hydrolysate.