Protein Sequence Determination

"All the King's horses and all the King's mm couldn't put Humpty Dumpty together again." It is entirely possible that the difficulty facing "all the King's men" was principally the lack of a sufficient guide to the techniques of reassembling from a series of small components, the original entity. It is the sincere hope of the editor of the present work and of each of the contributing authors that the modern researcher will not face a similar predicament in his endeavours to reconstruct the complete primary sequence of a protein from the array of component amino acids. Rather, it is the intent that, with this volume, he may proceed untimorously if not with outright confidence toward achieving his purpose. To the newcomer in protein sequencing, compelled by necessity, or fascination, to investigate the exact order of amino acids in proteins, the question of "where to begin" - or "how to do it" is urgent. To those more skilled, a ready source of additional techniques should nevertheless be of value. This volume attempts to present in a single source a discussion of the methods and techniques useful to the determination of the primary structures of proteins and peptides. Hopefully, this book will tell the reader "how to do it".

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.

Determination of the protein sequence is as important today as it was a half century ago, even though the techniques and purposes have changed over time. Mass spectrometry has continued its recent rapid development to find notable application in the characterization of small amounts of protein, for example, in the field of proteomics. The “traditional” chemical N-terminal sequencing is still of great value in quality assurance of the increasing number of biopharmaceuticals that are to be found in the clinic, checking processing events of recombinant proteins, and so on. It is joined in the armory of me- ods of protein analysis by such techniques as C-terminal sequencing and amino acid analysis. These methods are continually developing. The first edition of Protein Sequencing Protocols was a “snapshot” of methods in use in protein biochemistry laboratories at the time, and this, the second edition, is likewise. Methods have evolved in the intervening period, and the content of this book has similarly changed, the content of some chapters having been superceded and replaced by other approaches. Thus, in this edition, there is inclusion of approaches to validation of methods for quality assurance work, reflecting the current importance of biopharmaceuticals, and also a guide to further analysis of protein sequence information, acknowledging the importance of bioinformatics.

Sequence data--either lists of nucleotides or of amino acids--are now easily gathered using automated equipment; the real effort is involved in interpreting the data to produce predictions of protein structure or function. With the advent of worldwide computer networks, a plethora of software is now available for sequence analysis. This book describes the techniques for computer analysis of sequence data, with the emphasis on general issues rather than specific algorithms. Unlike many books on these topics, which focus on the "how-to" aspects of software packages, this one places more emphasis on the science behind the packages and on interpretation of the results.

How to design, execute, and interpret experiments for protein sequencing using mass spectrometry The rapid expansion of searchable protein and DNA databases in recent years has triggered an explosive growth in the application of mass spectrometry to protein sequencing. This timely and authoritative book provides professionals and scientists in biotechnology research with complete coverage of procedures for analyzing protein sequences by mass spectrometry, including step-by-step guidelines for sample preparation, analysis, and data interpretation. Michael Kinter and Nicholas Sherman present their own high-quality, laboratory-tested protocols for the analysis of a wide variety of samples, demonstrating how to carry out specific experiments and obtain fast, reliable results with a 99% success rate. Readers will get sufficient experimental detail to apply in their own laboratories, learn about the proper selection and operation of instruments, and gain essential insight into the fundamental principles of mass spectrometry and protein sequencing. Coverage includes: * Peptide fragmentation and interpretation of product ion spectra * Basic polyacrylamide gel electrophoresis * Preparation of protein digests for sequencing experiments * Mass spectrometric analysis using capillary liquid chromatography * Techniques for protein identification by database searches * Characterization of modified peptides using tandem mass spectrometry And much more

This book is an attempt to provide in a single source current state-of-the-art methodologies for protein sequence analysis. It is hoped that these various chapters are presented in such a way that both the newcomer and the established protein chemist will find useful information and directions to new techniques. This book offers a rich array of techniques and methods for sequencing proteins and peptides. It should meet the expectations of investigators in protein chemistry who wish to update their knowledge of sequencing techniques, and of those who wish to reacquaint themselves with the best available current technologies.

This book explores the remarkable information correspondences and probability structures of proteins. Correspondences are pervasive in biochemistry and bioinformatics: proteins share homologies, folding patterns, and mechanisms. Probability structures are just as paramount: folded state graphics reflect Angstrom-scale maps of electron density. The author explores protein sequences (primary structures), both individually and in sets (systems) with the help of probability and information tools. This perspective will enhance the reader’s knowledge of how an important class of molecules is designed and put to task in natural systems, and how we can approach class members in hands-on ways.