Purification of Extracted Peptides for Structural Analysis

The continued modifications in peptide/protein sequencer hardware, derivatization and coupling chemistry, reagent delivery, and component detection provide the protein/peptide chemist with the tools to determine primary structural information on subpicomole quantities of material. This dramatic quantum leap in sequencer sensitivity has made the preparation of samples even more critical with respect to purity. The task of isolating several hundred femtomoles or a few picomoles of a peptide of interest from a crude extract of cells or tissues seems daunting at first but is readily achievable if several important criteria are met and the requisite chromatographic hardware is available. A critical factor in initiating an isolation is the availability of a detection system for the peptide of interest. This may be a bioassay when the peptide of interest displays a novel biological activity, an immunoassay when the peptide displays cross-reactivity with antisera generated to a known peptide, or a chemical assay when structural attributes such as the presence of a C-terminal amide, sulfhydryl-containing, or aromatic residues are determined. Each of these detection systems has inherent advantages and disadvantages but, when possible, the system employed should be rapid and discriminating. Once a detection system has been chosen, and often the choice is dictated and limited by the individual study, a suitable quantity of starting material should be amassed and stored under conditions in which the peptide of interest is stable. The quantities required are a reflection of the relative abundance of the component of interest, but often the detection system employed plays a key role in determining this amount. Generally, bioassays require more starting material than chemical assays, which in turn require more than immunoassay. The value of performing pilot studies to determine the relevant detection system, quantity of starting material required, and optimal extraction medium to be employed should be stressed at this point. Once these parameters have been determined, the isolation procedure can be initiated. Where large quantities of tissues or cells are required, the initial extract, once tissue or cell debris has been removed by centrifugation, is often of considerable volume, may contain a high concentration of organic solvent, and is often turbid owing to the presence of microparticulates. If of an aqueous nature, the overall volume of the crude extract can be significantly reduced by lyophilization, but this procedure often incurs a large loss of peptide. The method of choice with such extracts would be, where appropriate, acidification followed by high-speed centrifugation to pellet microparticulates. Peptides present in the resultant clarified supernatant can be concentrated using disposable solid-phase extraction cartridges arranged in series, the number being related to the volume or density of the extract. An extract containing organic solvent can be treated in a similar manner after removal of solvent by prior rotary evaporation. The cartridges can be eluted step-wise with ascending concentrations of acetonitrile and each eluate can be assayed for the peptide of interest. The preparation of neuropeptide-containing fractions from tissues is dealt with in Chapter 1 .