Nanopreparative purification of peptides and proteins by HPLC

In order to take full advantage of the sensitivity of modern protein characterization techniques, such as amino acid composition and sequence analysis, fairly pure samples of a single polypeptide are usually required. Although HPLC has been used extensively for isolation and purification of peptides and proteins at levels above one microgram, problems with sensitivity, resolution and recovery are often encountered when isolating nanogram (low picomole) levels of a polypeptide from a complex sample. This paper explores the requirements for nanopreparative purification of polypeptides by HPLC and details the design of a microbore HPLC system which allows reproducible, high sensitivity, high resolution separations of peptides and proteins at the low picomole level. The application of this microbore HPLC system to the isolation and characterization of a trace variant from a partially purified recombinant protein sample is also detailed.