PURIFICATION OF PROTEINS AND PEPTIDES FOR SEQUENCE-ANALYSIS USING MICROCOLUMN LIQUID-CHROMATOGRAPHY

Reversed phase microcolumn (0.32 mm i.d.) liquid chromatography was used to purify low-picomole amounts of proteins and peptides suitable for structural analysis. Using this approach, rapid trace enrichment (concentration) of low nanogram levels of proteins from volumes as high as 500 muL down to 1-2 muL was demonstrated. The total system recovery (including manual collection and reinjection) for 50 ng of lysozyme was >95%; the overall recovery after five injections was 90%. Using the same packing, Brownlee RP-300, the resolution of a standard mixture of proteins was comparable for columns varying in length from 250 mm to 10 mm. Identification by amino acid sequence analysis of selected peptides recovered from a sub-10 pmol Staphylococcus aureus V8 protease digest of recombinant murine interleukin-6 (IL-6) was achieved.