HPLC of Peptides and Proteins: Methods and ProtocolsMarie-Isabel Aguilar 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. |
Contents
Immobilized Metal Ion Affinity Chromatography of Proteins | 89 |
Immunoaffinity Chromatography of Proteins | 103 |
Liquid ChromatographyMass Spectrometry and Tandem | 111 |
Capillary Separations | 143 |
Micropreparative HPLC of Peptides and Proteins | 165 |
Nice and MarieIsabel Aguilar | 177 |
Analytical HighPerformance Liquid Chromatography | 183 |
PrepSemiprep Separations of Peptides | 191 |
HPLC in the Analysis of Peptide Metabolism | 275 |
Isolation and Characterization of Naturally Processed | 291 |
Australian FunnelWeb Spider Venom Analyzed | 307 |
HPLC and Mass Spectrometry of Intrinsic Membrane | 323 |
IgG Purification | 341 |
LCMS to Identify Key Domains | 351 |
Sensitive Enzymatic Analysis of Histidine Decarboxylase | 365 |
Automated vs Manual Profiling of Peptide Libraries | 377 |
Other editions - View all
Common terms and phrases
acetic acid acetonitrile aliquot Amersham Biosciences amino acid ammonium ammonium bicarbonate analysis analytical antibody Approx binding biological bradykinin buffer capillary capLC cell Chem chro Chromatogr cleavage column volumes concentration containing Crosslinked detection detector digestion electrospray electrospray ionization eluent elution enzyme equilibration filter flow rate formic acid fractions histamine histidine HPLC HPLC of Peptides hydrophilic hydrophobic IMAC imidazole immobilized inhibitors injection interactions ion-exchange ionization LC-MS LC–MS ligand liquid chromatography loading matrix-assisted laser desorption/ionization membrane methanol Methods and Protocols Milli-Q mL/min mobile phase mode molecular molecules MS/MS NaCl Note organic solvent packed particle peak peptide mapping peptides and proteins performed phosphorylated preparative proteomics pump purification quadrupole residues resin resolution retention RP-HPLC salt sample scan separation sequence silica sodium solution solvent spectra spectrum stationary phase Subheading tandem mass spectrometry techniques temperature tion Tris-HCl trypsin tryptic tubing venom Wash