Advanced Methods in Protein Microsequence Analysis

Much of the recent spectacular progress in the biological sciences can be at­ tributed ot the ability to isolate, analyze, and structurally characterize proteins and peptides which are present in cells and cellular organelles in only very small amounts. Recent advances in protein chemistry and in particular the application of new micromethods have led to fruitful advances in the understanding of basic cellular processes. Areas where protein-chemical studies have resulted in interest­ ing discoveries include the peptide hormones and their release factors, growth factors and oncogenes, bioenergetics, proton pumps and ion pumps and chan­ nels, topogenesis and protein secretion, molecular virology and immunology, membrane protein analysis, and receptor research. In fact, the key methods are now on hand to unravel many of the major outstanding problems of molecular biology and in particular questions of fundamental interest which relate to devel­ opmental biology and specificity in cell-cell interaction. In this volume we have assembled descriptions of procedures which have re­ cently been shown to be efficaceous for the isolation, purification, and chemical characterization of proteins and peptides that are only available in minute amounts. Emphasis is placed on well-established micromethods which have been tested and found useful in many laboratories by experienced investigators. The chapters are written by specialists, and describe a range of sensitive techniques which can be used by researchers working in laboratories with only modest resources and equipment.

1 Separation and Amino Acid Analysis of Proteins and Peptides for Microsequencing Studies.- 1.1 Separation of Peptides.- 1.2 High Performance Liquid Chromatography of Proteins.- 1.3 Two-Dimensional Polyacrylamide Gel Electrophoresis in Stamp-Sized Gels.- 1.4 Amino Acid Analysis by High Performance Liquid Chromatography of Phenylthiocarbamyl Derivatives.- 1.5 High Sensitivity Amino Acid Analysis Using DABS-C1 Precolumn Derivatization Method.- 2 Manual and Solid-Phase Microsequencing Methods.- 2.1 Modern Manual Microsequencing Methods.- 2.2 Manual Microsequence Determination of Proteins and Peptides with the DABITC/PITC Method.- 2.3 Solid-Phase Microsequencing: Procedures and Their Potential for Practical Sequence Analysis.- 2.4 Automated Solid-Phase Microsequencing Using DABITC, On-Column Immobilization of Proteins.- 3 Gas-Phase and Radio-Sequence Analysis.- 3.1 Gas-Phase Sequencing of Peptides and Proteins.- 3.2 Water Contents and Quality Criteria of Microsequencing Chemicals. Preliminary Results of a Reevaluation.- 3.3 An Improved Gas-Phase Sequenator Including On-Line Identification of PTH Amino Acids.- 3.4 Amino-Acid Composition and Gas-Phase Sequence Analysis of Proteins and Peptides from Glass Fiber and Nitrocellulose Membrane Electro-Blots.- 3.5 Protein Blotting from Polyacrylamide Gels on Glass Microfiber Sheets: Acid Hydrolysis and Gas-Phase Sequencing of Glass-Fiber Immobilized Proteins.- 3.6 Radio-Sequence Analysis; an Ultra-Sensitive Method to Align Protein and Nucleotide Sequences.- 4 Phenylthiohydantoin Identification, On-Line Detection, Sequences Control, and Data Processing.- 4.1 Conversion of Anilinothiazolinone to Phenylthiohydantoin Derivatives and Their Separation by High Pressure Liquid Chromatography.- 4.2 The Use of On-Line High Performance Liquid Chromatography for Phenylthiohydantoin Amino-Acid Identification.- 4.3 Device Control, Data Collection, and Processing in Protein Micro-Sequencing and Amino-Acid Analysis.- 5 Analysis of Cysteine Residues in Proteins.- 5.1 Analysis of Cyst(e)ine Residues, Disulfide Bridges, and Sulfhydryl Groups in Proteins.- 5.2 Identification of the Heme-Binding Cysteines in Cytochromes c Without Radioactive Labeling.- 5.3 Micro-Isolation of Polypeptides Precolumn Labeled with Hydrophobic Chromophore.- 6 Methods of Analyzing Protein Conformation.- 6.1 Synthetic Immunogens for Secondary Structure Assignment: Conformational Sequencing of Proteins with Antipeptide Antibodies.- 6.2 Aspartyl-tRNA Synthetase-Induced Aspartylation of Proteins: a Fingerprint Approach to Map Accessible Domains in Protein.- 7 Strategies and Specific Examples of Sequencing Proteins and Peptides.- 7.1 Strategies of Biochemical Characterization of Hormonal Peptides.- 7.2 Use of a Metalloproteinase Specific for the Amino Side of Asp in Protein Sequencing.- 7.3 Primary Structure of Rabbit Apolipoprotein A-I. High Performance Liquid Chromatography, PTC-Amino Acid Analysis, and Microsequencing.- 7.4 Sequence Analysis of Complex Membrane Proteins (Cytochrome c Oxidase).- 7.5 Sequence Studies on the ?-, ?-, and ?-Chains of Elongation Factor 1 from Artemia. Some Practical Notes.- 8 Alternative Sequencing Techniques for Elucidation of Peptide and Protein Structures.- 8.1 RNA Sequencing.- 8.2 Synthetic Genes as a Powerful Tool for Protein Structure-Function Analysis.- 8.3 Generation and Interpretation of Fast Atom Bombardment Mass Spectra of Modified Peptides.