Peptide Synthesis

Peptides are synthesized utilizing the FMOC chemistry and is performed using an automated Applied Biosystems 432A peptide synthesizer, also known as the Synergy.

Method: The Facility operates an Applied Biosystems 432A peptide synthesizer. This instrument combines the FMOC chemistry with the HBTU activation to perform solid-phase peptide synthesis. Synthesis starts with the C-terminal amino acid. Amino acids are then added one at a time till the N-terminus is reached. Three steps are repeated each time an amino acid is added. They are: (1) Deprotection of the N-terminal amino acid of the peptide bound to the resin. (2) Activation and addition of the next amino acid. (3) Deprotection of the new N-terminal amino acid. Of course, there are wash steps in between. The synthesizer is capable of monitoring the deprotection and coupling steps. The printouts of the monitored steps are our first level of quality control.

Post-synthesis procedures and Quality Control: At the end of the synthesis the protected peptide and the resin are collected. The next step is to cleave the peptide from the resin and to remove the side-chain protection groups from the peptide. Both cleavage and deprotection are carried out in the presence of 90% TFA, 5% thioanisole and 2.5% ethanedithiol. After separating the resin by filtration through glass wool, the peptide is precipitated in the presence of MTBE (methyl t-butyl ether). Diethyl ether is used in the case of very hydrophobic peptides. The peptide is washed 3 times with MTBE in order to remove the protection groups and to neutralize any leftover acidity. The purity of the peptide is further monitored by mass spectrometry and in some case by amino acid analysis and sequencing.

Scale of synthesis: All amino acids come prepackaged at 25 µmol. For a 20 amino acid peptide, for example, one should roughly expect about 40 mg of peptide. However, due to the number of steps involved including the chemistry, cleavage and deprotection, and extraction and washing the final recovery of the peptide is in the range 25-30 mg which is enough for most purposes.

Peptide modifications: All what is described in this paragraph is carried out on the synthesizer with very minor interventions. An amide could be added at the C-terminus of the peptide. An acetyl group could be added to the N-terminus. Biotin, stearate and other modifications could be added to the N-terminus. Other modifications are also possible (please inquire).

Data Provided: Copy of the mass profile and the request form. Should amino acid sequencing be performed a data sheet of the sequence information will be supplied. The actual sequence chromatograms will be stored in the facility and are available upon request.

Turn-around time: Normally for a single peptide the turn around time is 4-5 working days. Additional peptides will require 1-2 day per peptides.

modifications $25/peptidemodifications $50/peptide

Cost:	on-campus:	$20.00/amino acid
	off-campus:	$25.00/sample