Instrumentation

Liquid chromatography is used extensively to purify proteins and peptides from mixtures of natural materials. We routinely use reverse phase chromatography to analyze peptides and isolate proteolytic fragments of proteins for subsequent amino acid sequence analysis or compositional analysis. The Laboratory is equipped with two high-pressure liquid chromatography systems for protein and peptide purification. A Waters 626 system composed of a model 600 system controller and 486 variable wavelength detector. The system is controlled by Millenium32 software that is also used for data analysis. The system, used for analytical and semi-preparative applications, is constructed of PEEK materials and is compatible with biological samples in addition to organic solvents routinely used for reverse phase chromatography. A SuperLoop attachment along with the ability to mix solvents from four separate reservoirs enables us to use the instrument in a "scouting" mode to optimize separation conditions for biological samples using aqueous-based . A stand-alone Hewlett Packard 1100 HPLC system is equipped with an autosampler and a diode array detector (DAD) for spectral analysis and is used for narrowbore, microbore and capillary LC peptide mapping and purification using reverse phase chromatographic techniques. This instrument is used in conjunction with the MALDI-TOF and Hewlett Packard G1005A Protein Sequencer to obtain internal protein sequence information from protein digests that can allow investigators to identify their proteins of interest and aid in the design of

the appropriate probes for their isolation and, possible, cloning. An LC Packings Probot is attached to the HP1100 HPLC to aid in automated spotting of peptides to a sample plate for MALDI-TOF mass spectrometry (see below). In addition, the facility has a Pharmacia Äkta Explorer 10 Protein Purification system that is capable of running aqueous and organic mobile phases for native protein purification as well as peptide isolation. This instrument is being used to develop pilot protein purification protocols using a library of columns that includes hydrophobic interaction, ion exchange, metal chelation and affinity matrices.