HPLC of Peptides and Proteins

The introduction of high-performance liquid chromatography (HPLC) to the analysis of peptides and proteins some 25 years ago revolutionized the biological sciences by enabling the rapid and sensitive analysis of peptide and protein structure through the exquisite speed, sensitivity, and resolution that can be easily obtained. Today, HPLC in its various modes has become the pivotal technique in the characterization of peptides and proteins and currently plays a critical role in both our understanding of biological processes and in the development of peptide- and protein-based pharmaceuticals. The number of applications of HPLC in peptide and protein purification continues to expand at an extremely rapid rate. Solid-phase peptide synthesis and recombinant DNA techniques have allowed the production of large quantities of peptides and proteins that need to be highly purified. HPLC techniques are also used extensively in the isolation and characterization of novel proteins that will become increasingly important in the postgenomic age. The design of multidimensional purification schemes to achieve high levels of product purity further demonstrates the power of HPLC techniques not only in the characterization of cellular events, but also in the production of pepti- and protein-based therapeutics. HPLC continues to be at the heart of the analytical techniques with which scientists in both academia and in industry must arm themselves to be able to fully characterize the identity, purity, and potency of peptides and proteins.

Includes supplementary material: sn.pub/extras

Klappentext
High performance liquid chromatography (HPLC) plays a critical role today in both our understanding of biological processes and in the development of peptide and protein-based pharmaceuticals. In HPLC of Peptides and Proteins: Methods and Protocols, leading experts from academia and industry comprehensively describe how to successfully perform all the critical HPLC techniques needed for the analysis of peptides and proteins. The methods range from commonly used techniques to those for capillary to large-scale preparative isolation. The authors have also presented a number of specific applications as case studies to illustrate the analytical approaches to a particular separation or assay challenge, with examples drawn from contemporary fields in biochemistry and biotechnology. These applications include proteolytic mapping, posttranslational modification, neuropeptide processing, glycopeptides and glycoproteins, MHC-binding peptides, toxins/venoms, membrane proteins, antibodies, combinatorial and proteome analysis, and enzymatic activity. Each readily reproducible protocol contains background notes, step-by-step instructions, reagent and equipment lists, and tips on both troubleshooting and avoiding known pitfalls.
Comprehensive and easy-to-follow, HPLC of Peptides and Proteins: Methods and Protocols offers every biologist and biotechnologist at all levels of expertise ready access to the powerful tools needed to develop and carry out peptide and protein separations and analyses today.

Inhalt
Modes of HPLC.- HPLC of Peptides and Proteins.- Reversed-Phase High-Performance Liquid Chromatography.- Ion-Exchange Chromatography.- High-Performance Hydrophobic Interaction Chromatography.- Gel Filtration Chromatography.- Hydrophilic Interaction Chromatography.- Immobilized Metal Ion Affinity Chromatography of Proteins.- Immunoaffinity Chromatography of Proteins.- Liquid Chromatography-Mass Spectrometry and Tandem Mass Spectrometry of Peptides and Proteins.- Preparative Methodologies.- Capillary Separations.- Micropreparative HPLC of Peptides and Proteins.- Multidimensional HPLC Purification of Proteins.- Analytical High-Performance Liquid Chromatography.- Prep/Semiprep Separations of Peptides.- Large-Scale Protein Chromatography.- Applications.- Proteolytic Peptide Mapping.- Mass Spectrometric Characterization of Posttranslationally Modified ProteinsPhosphorylation.- Analyses of Glycopeptides and Glycoproteins by Liquid Chromatography-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry.- HPLC in the Analysis of Peptide Metabolism.- Isolation and Characterization of Naturally Processed MHC-Bound Peptides From the Surface of Antigen-Presenting Cells.- Australian Funnel-Web Spider Venom Analyzed With On-Line RP-HPLC Techniques.- HPLC and Mass Spectrometry of Intrinsic Membrane Proteins.- IgG Purification.- DNA-Binding Proteins.- Sensitive Enzymatic Analysis of Histidine Decarboxylase Using HPLC.- Automated vs Manual Profiling of Peptide Libraries by Mass Spectrometry.- Proteome Analysis.