Within the PPMC, many different syntheses have been performed over the years. This has included various bundlemer peptide sequences, as well as other peptide sequences. Additionally, there have been a variety of chemical reactions utilized for connecting and/or removing reactive handles to and from peptides. Syntheses and chemical reactions that have protocols that have been optimized are linked below, as well as suggested peptide synthesis workflows. The procedures included have been found to be generally applicable, but they may not be optimal in every synthesis. Various factors, including production scale, peptide sequence, and peptide length, may require modification of these procedures for best results. The following Table of Contents links to different information related to the peptide synthesis workflow and protocols for peptide modifications. Click on the associated links to visit those pages. Feel free to learn more about the overall workflow by continuing to read below the Table of Contents.
Table of Contents
Solid-Phase Peptide Synthesis (SPPS)
Instrumentation:
CEM Liberty Blue Peptide Synthesizer
Gyros Protein Technologies PurePep Chorus Peptide Synthesizer
On-Resin Modifications
N-Terminus Acetylation Protocol
Alloc Protecting Group Removal Protocol
Maleimide/Carboxylic Acid Coupling Protocol
Removing Peptide from Resin
Peptide Cleavage from Resin Protocol
Purification: Reversed-Phase High Performance Liquid Chromatography (RP-HPLC)
Instrumentation:
Lyophilization
Instrumentation:
Labconco FreeZone 12 Liter -84C Console Freeze Dryer
SP Scientific Genevac EZ-2 Elite Centrifugal Evaporator
Characterization
Instrumentation:
Jasco J-1500 Circular Dichroism Spectrophotometer
Automated Circular Dichroism (CD) Data Analysis
Scientific Resources – Circular Dichroism (CD)
Microcleavage and Mass Spectrometry Characterization
Peptide Synthesis Workflow
The steps taken for obtaining pure peptide are detailed above. This first involves Solid-Phase Peptide Synthesis (SPPS) using one of two PPMC automated peptide synthesizers: CEM Liberty Blue Peptide Synthesizer, or Gyros Protein Technologies PurePep Chorus Peptide Synthesizer. For more information about the basics of SPPS, see the associated page.
Once a peptide has been synthesized on resin, many different modifications can be made to the peptide. This includes modifications to the N-terminus, such as acetylation, as well as the addition of click reactive handles like maleimide or azide functional groups, that can be utilized for polymerization reactions. There are also options to use special protecting groups on side chains of internal amino acid residues for side-chain modifications. Protocols for N-terminus and side-chain modifications are linked in the Table of Contents at the top of the page. After modifications have been performed, the peptide is cleaved from resin. Check out our Peptide Cleavage from Resin Protocol.
Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) is then used to purify the peptide from organic scavengers, terminated sequences, deletion sequences, and chemically modified sequences from side reactions during either the synthesis or the cleavage of the peptide from resin. This highly selective method allows users to obtain peptide products greater than 95% pure. Within the PPMC, we have access to a Teledyne ISCO ACCQPrep HPLC, which allows for automated fraction collection. For more information about the basics of RP-HPLC, see the associated page.
Once a peptide has been purified, it is then lyophilized utilizing either our traditional Labconco Lyophilizer or our Genevac Centrifugal Evaporator. This removes the water from the material to stabilize the peptide for freezer storage. Once this step is completed, the peptide is ready to be used for the desired applications. For more information on lyophilization of peptides, see the associated page.
Peptides that result from SPPS are lyophilized trifluoroacetate (TFA) salts. For applications where residual TFA is undesirable, TFA can be exchanged for other ions and can be confirmed using 19F-Nuclear Magnetic Resonance (NMR) Spectroscopy. For peptides that form secondary structures, Circular Dichroism (CD) Spectroscopy is an easy way to evaluate a peptides folding characteristics. Within the PPMC, we have access to a Jasco J-1500 Circular Dichroism Spectrophotometer. For more information about the technique of circular dichroism spectroscopy, see the associated page.
Throughout the peptide synthesis process, Mass Spectrometry Characterization is also performed. This allows scientists to ensure the desired product is properly synthesized.