Reverse-phase high-performance liquid chromatography of Escherichia coli ribosomal small subunit proteins

Reverse-phase high-performance liquid chromatography has been explored as an approach for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes. The majority of these proteins are of similar molecular weight and isoelectric point, making separation by size exclusion or ion exchange difficult. With the use of an octadecasilyl silica column and a trifluoroacetic acid-acetonitrile solvent system, the 21 proteins of the 30 S subunit have been separated into 15 peaks. The yield of total protein recovered from the column was ≥85%. The proteins present in each peak have been identified by polyacrylamide gel electrophoretic analysis of the peaks as well as by comparison with the relative retention volumes of known purified 30 S proteins on the column. The results clearly show that this method is a powerful and rapid technique for the identification and purification of 30 S proteins. Analysis of ³H]puromycin-labeled 30 S subunit protein provides an illustrative example of its utility for affinity labeling studies.