Direct Micro-Sequence Analysis of Peptides fromEscherichia coliRibosomal Proteins S11, L9 and L29 after Separation by Reversed Phase Chromatography

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Abstract
Tryptic peptides of the ribosomal proteins S11, L9 and L29 were separated by reversed phase chromatography under conditions which enabled direct micro-sequencing with the 4-(dimethylamino)azobenzene-4''-isothiocyanate/phenylisothiocyanate double coupling method. The peptides were separated on a RP-18 column employing volatile buffers at pH 2.0, 4.1 and 7.8. Depending on the different chromatographic behavior of the peptide mixture, the elution gradient was optimized for each hydrolysate using 20 .mu.g of the hydrolyzed protein. Preparative separations were made with 150-250 .mu.g. At least 80% of the peptides could be isolated by these techniques and used for direct micro-sequencing without further purification of desalting. The high performance liquid chromatographic method employed allows easy isolation and sequencing with minute amounts of peptides.