We are using a Protein Technologies Inc., model Tribute peptide synthesizer with on-line UV monitoring of FMOC deprotection and conditional cycle chemistry feedback. This instrument offers three scales of synthesis: 0.1, 0.3 and 0.5 mmol. Starting with polystyrene or tentagel resin, the peptide is built "backwards," from the C-terminus to the N-terminus, one amino acid at a time. The peptide is always written in N to C terminal format just as DNA is assumed to be written in 5’ to 3’ format. After synthesis, the peptide is cleaved from the resin, yielding the crude form of the peptide. Generally, this type of synthesis yields crude peptides with 70-85% purity, but HPLC purification can be done if higher purity is desired. Typical peptide lengths are 5-40 amino acids. Our longest to date is a 72 mer.
Certain combinations of amino acids cause aggregation and result in low coupling yields and poor quality peptides. We may reduce the scale somewhat for peptides with long and/or difficult sequences. Usually these sequences occur in the 7 to 15 amino acid stretch emanating from the C-terminal. Di-peptides or pseudoprolines may be incorporated to increase yields if a user desires but these reagents are relatively expensive and we must pass this cost along to you. Some difficult sequences are obvious to people who have peptide synthesis experience, but most are not. Because the rate at which FMOC deprotection occurs is proportional to the rate at which the incoming residue will couple, UV on-line monitoring of the FMOC cation released after N-terminal deprotection is crucial for the synthesis of long peptides. This synthesizer can determine when aggregation occurs and modify reaction vessel conditions to compensate by extending the deprotection and coupling periods. This results in high quality short and long peptides.
The peptide, in both crude and purified forms, is given out as dry powder with a specified weight. HPLC and mass spectrometry data are provided as well.
To determine how much peptide is present:
- Assume that about 75-80% by weight is peptide, with the remainder being H20 and TFA salt
- If there is Tyr, Trp, Phe, or Cys in the sequence, you can use the extinction coefficients and Beer’s Law for those amino acids to calculate the amount present. (if monitoring at 280 nm, eY=1280, eW=5690, eC=120, eF=2 e=extinction coefficient in units of Lmol-1cm-1)
epeptide=aeY + beW + ceC + deF, where a=# Y in sequence, b=#W, c=#C, d=#F Use c=A/(epeptide*l), where c=molar concentration of peptide in mol/L, A=absorbance at 280nm, and l=path length of cuvette in cm
Alternatively, Google for “Spectrophotometric Determination of Protein Concentration” and find an excellent explanation in Current Protocols in Protein Science.
- Amino acid analysis is the most accurate method. We did this here for years and can help you interpret the AAA data if you like. AAA allows you to use each residue to quantify the peptide which allows you to average out individual residue arrors. We suggest going to the ABRF Core Market Place and selecting “amino acid analysis” to get a list of academic and commercial labs that perform amino acid analysis. Alternatively try Googling “who does amino acid analysis”!