Advanced Methods in Protein Sequence Determination

Confusion now hath made his masterpiece Macbeth II iii 72 Whence and what are those execrable shape? Paradise Lost Ib 1 681 Confusion worse confounded Paradise Lost Ib 1 995 When the manuscript for the first part of this book was proposed, it was anticipated that the discussion of the entire field of protein se­ quencing could be covered in a single volume - from purification and characterization of the protein through fragmentation by chemical or enzymic means and, finally, to reassembly of the identified individual peptides into the reconstructed total sequence. It soon became evident that this would not be possible. While the intent was to restrict the expose of procedures only to that information concerned with "hands on" wet chemistry, it became apparent that a thorough presentation would require, in addition, a discussion of certain instrumental and more theoretical approaches not included in the first volume. Furthermore, the entire understanding of the field of protein sequencing has advanced appreciably since the inception of this book. The purpose of the first volume was to provide practical information in sufficient detail to permit the researcher to undertake the actual sequencing procedures in his own laboratory.

1 Step-wise Degradation of Peptides Attached to Solid Supports.- I. Introduction.- II. Choice of Solid Support.- III. Attachment of Peptides to Supports.- IV. Step-wise Degradation Procedures.- V. Automation of Solid Phase Degradation.- VI. Concluding Remarks.- 2 Coupling Methods and Strategies in Solid-Phase Sequencing.- I. Introduction.- II. Coupling Procedures.- III. Sequencing Strategies.- 3 Sequencing Peptides and Proteins Lacking Free ?-Amino Groups.- I. Introduction.- II. Isolation and Detection of Peptides Lacking Free ?-Amino Groups.- III. PCA-Terminating Peptides and Proteins.- IV. Acylated Peptides and Proteins.- V. Concluding Remarks.- 4 Use of Antibody in the Study of Protein Structure.- I. Introduction.- II. Measurement of the Ag-Ab Reaction.- III. The Effect of Conformational Changes on the Antigenicity of Proteins.- IV. Detection of Sequence Changes in Proteins with Antibodies.- V. Use of Antibody in the Study of Protein Evolution.- 5 Polarization of Light and Protein Structure.- I. Introduction.- II. Plane Polarized Light.- III. Circularly Polarized Light.- IV. Circular Dichroism.- V. Drude Equation.- VI. Optical Properties of Amino Acids.- VII. Optical Properties of Peptides.- VIII. Conclusion.- 6 The X-ray Crystallography Technique in Protein Sequencing.- I. Introduction.- II. Outline of the Protein Crystallographic Method.- III. Some Important Concepts in X-ray Diffraction Analysis of Protein Crystals.- IV. Building up the Electron Density Map.- V. Interpretation of the Electron Density Map.- VI. Correlation of the Chemical Sequence with the Electron Density Maps.- VII. Conclusion.- 7 Amino Acid Sequence Determination by Mass Spectrometry.- I. Introduction.- II. Instrumentation and Techniques.- III. Mass Spectrometry of Peptides.- IV. ConcludingRemarks.- 8 Peptide Sequence Analysis by Nuclear Magnetic Resonance Spectroscopy.- I. Introduction.- II. NMR Principles.- III. Amino Acid Composition and Residue Identification.- IV. Conclusions.- References.