Methods of Protein Microcharacterization

Milestones in the techniques and methodology of polypeptide structure determination include the determination of the sequence of insulin by Sanger in 1951 (I) and the introduction of the repeti tive degradation of proteins with phenylisothiocyanate by Edman in 1959 (2). The automation of Edman chemistry (3) played a major role in the determination of polypeptide structures. Important modifications of Edman chemistry include the solid-phase approach by Laursen in 1971 (4) and the use of modified Edman reagents such as 4-N,N-dimethylaminoazobenzene-4'-isothiocy- ate (DABITC) for manual sequencing by Chang et al. (5) in 1976. A second major breakthrough in the analysis of polypeptides was automated amino acid analysis described by Spackman et al. in 1958 (6). However, during the period from 1975 to 1980, it became increasingly clear that the amount of material required for struc tural analysis was more than could be easily isolated for the vast majority of proteins. The field was criticized for its lack of sensitive techniques for the analysis of growth factors, immune modulators, membrane receptors, and peptide hormones. In addition, very little had been done to modernize and improve the original instruments introduced in the mid-1960s. The first indications of improved instrumentation for Edman chemistry came from Wittmann-Liebold's laboratory (7), followed by the introduction of a "micro" sequencer by Hunkapiller and Hood in 1978 (8). The movement toward improved instrumentation culminated in the "gas"-phase sequencer of Hewick et al. (9) in 1981.

Contenu
Microisolation Techniques.- Microisolation of Biologically Active Polypeptides by Reverse-Phase Liquid Chromatography.- Reverse-Phase HPLC Isolation and Microsequence Analysis.- Purification of Microgram Quantities of Proteins by Polyacrylamide Gel Electrophoresis.- Microamino Acid Analysis.- Amino Acid Analysis With Postcolumn Fluorescent Derivatization.- Amino Acid Analysis by o-Phthaldialdehyde Precolumn Derivatization and Reverse-Phase HPLC.- Amino Terminal Analysis.- Manual Edman Sequencing System.- Comparison of the Spinning Cup and Gas-Phase Instruments.- Gas-Phase Protein/Peptide Sequencer.- Design of a Multipurpose Sequencer.- Solid-Phase Methods in Protein Microsequence Analysis.- Analysis of Phenylthiohydantoin Amino Acids by HPLC.- Purification and Preparation of Chemicals for Microsequencing With the Edman Degradation.- Carboxyl-Terminal Analysis.- Microsequence Analysis by Enzymatic Methods.- COOH-Terminal Analysis of Polypeptides by Chemical and Chromatographic Methods.-Mass Spectrometric Analysis of Polypeptides.- Polypeptide Sequence Analysis Using Gas Chromatography-Mass Spectrometry.- Fast Atom Bombardment and Secondary Ion Mass Spectrometry of Peptides and Proteins.