HPLC of Peptides and Proteins

Abstract

High-performance liquid chromatography (HPLC) is an essential tool for the purification and characterization of biomacromolecules. This unit presents a thorough discussion of the eight types of HPLC currently used, highlighting equipment and start-up procedures, recommendations for running each type of experiment, and theoretical considerations for the separation of peptides and proteins. This is an excellent primer for HPLC users.

New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications

“…Because of its versatility, flexibility and robustness, RPLC is one of the dominant approaches used for the analysis of peptides and proteins [11]. High-molecular-mass compounds such as intact proteins may have numerous different conformations, posttranslational modifications, or multiple isoforms that can cause broadened peak shapes and shifted retention times in the chromatograms.…”

“…For the separation of the diverse components of a sample that contains peptides or proteins of an unknown composition, an initial scouting gradient is typically run to determine the intervals of the variables to be optimized [11,17]. In many cases, the nature of the components is unknown.…”

Targeted Degradation of Proteins by PROTACs

“…Once the formation of the fully assembled PROTAC is confirmed, purify it using a normal-phase (silica-based column) HPLC (see, e.g., Boysen and Hearn, 2001). Once the formation of the fully assembled PROTAC is confirmed, purify it using a normal-phase (silica-based column) HPLC (see, e.g., Boysen and Hearn, 2001).…”

“…Therefore, it is recommended to prepare a target protein ligand motif and an E3 recognition motif independently, and to couple them in DMSO or DMF. Additional reagents and equipment for mass spectrometry (Carr and Annan, 1996) and HPLC (Boysen and Hearn, 2001) 1. Dissolve DHT-Gly-monosuccinimidyl suberate (1.5 equivalents) in anhydrous DMF.…”

“…Current Protocols in Chemical Biology Volume 2 8. Once the formation of the fully assembled PROTAC is confirmed, purify it using a normal-phase (silica-based column) HPLC (see, e.g., Boysen and Hearn, 2001). A gradient solvent system (hexane-isopropyl alcohol) for HPLC is recommended for best results.…”