HPLC analysis and purification of peptides.
High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptides/proteins by size-exclusion chromatography and monitoring the hydrophilicity/hydrophobicity of amphipathic alpha-helical peptides, a vital precursor for the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins.
Mant
Colin T
CT
Department of Biochemistry and Molecular Genetics, University of Colorado at Denver and Health Sciences Center, Aurora, CO, USA.
Chen
Yuxin
Y
Yan
Zhe
Z
Popa
Traian V
TV
Kovacs
James M
JM
Mills
Janine B
JB
Tripet
Brian P
BP
Hodges
Robert S
RS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D002852:Chromatography, Ion Exchange / Q000379:methods; D004355:Drug Stability; D019075:Electrophoresis, Capillary / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000592:standards; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012015:Reference Standards; D013696:Temperature
D000596:Amino Acids; D023181:Antimicrobial Cationic Peptides; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins