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FAQs

Technical

• What are pseudoproline amino acids and what advantage do they have in peptide synthesis?
• What are the typical contaminants associated with peptide synthesis?
• What is the difference between peptide purity and peptide yield?
• What are the typical salts associated with my peptide?
• If I have biotin in my peptide, do I need a spacer between the biotin group and the peptide sequence and if so which spacer should I use?
• What can I do to increase the cell permeability of a peptide?
• How do I solubilize my peptides?
• How do I characterize batches of my "drug candidate" peptide, which contains a D-amino acid?

  There are many analytical techniques available and we have listed below the most common ones, with some comments:

  1. Mass spectrometry (MS) is one of the most powerful techniques for checking that the "molecular formula" is correct, i.e. the peptide contains the stated amino acids. MS also can be used (on the right machine) for confirming the amino acid sequence of the peptide, by fragmenting the peptide within the instrument and analysing the fragments. This technique still does not readily distinguish stereoisomers of the "correct" sequence (in this case, enantiomeric peptides).
  2. Analytical reverse phase HPLC is a very sensitive technique for detecting differences in physicochemical properties between similar peptides, and has the potential to act as a quality check method for whether or not amino acids of the correct stereochemistry were used to make the peptide. It requires the use of well-characterized standards, or can be used to get a measure of "relative" properties (differences) between test samples. However, identical elution of two samples in analytical HPLC is, in itself, NOT proof that the samples are identical.
  3. High pressure liquid chromatography, combined with continuous mass spectrometry of the eluate from the chromatography column (i.e. LC/MS) combines the power of HPLC and MS to give a more detailed picture of the components of a mixture, but may not be useful if the material is homogeneous by HPLC.
  4. Polarimetry (optical rotation of polarized light) is a simple technique for verifying that the peptide has the expected optical properties (a function of its stereochemistry), but polarimetry may not have enough sensitivity to detect a change in a single amino acid.
  5. N- or C-terminal amino acid sequencing is a good way to ensure the amino acids were assembled in the correct order, but is rarely necessary because a similar assurance can be obtained from comparative analytical HPLC (by comparison with verified standards), fragmentation mass spectrometry, NMR etc.
  6. 6. Amino acid analysis is good for ensuring the correct amino acids are present, and in the expected ratios, and is also one of the best ways to measure the true "peptide content" (i.e. it does not measure counterions, solvent and nonpeptide impurities). It is not a good way to detect amino acids with incorrect stereochemistry (i.e. L- versus D- isomers).
  7. Depending on the size of the peptide, 2-D proton NMR may be suitable for setting an absolute characterization standard for your peptide, but it may require a lot of development work by a chemist to assign an identity to each signal, and therefore to confirm that the data shows the structure is exactly what you expected it to be.
  8. Chiral column HPLC is another way to separate peptides of identical amino acid sequence, differing in stereochemistry of one or more of the amino acids. A chiral column can also be used to check the stereochemistry of the individual amino acids after hydrolysis of the peptide.
  9. Capillary electrophoresis is a very sensitive technique for testing the homogeneity of a peptide, and could also pick up mistakes in the assembly/stereochemistry of the peptide, by comparison with verified standards.
  10. Other supplementary methods would include circular dichroism, bioactivity measurement, immunoreactivity (recognition by a specific antibody), elemental analysis, and spectrophotometry.

  Testing for impurities, in contrast to checking for identity, would make use of the techniques above in different ways. The presence of "unexpected" peaks or signals would be evidence for impurities; however, the absence of unexpected peaks or signals would not be proof of the absence of impurities. More content in preparation (check back soon), for example:

  • Isomerization
  • Bioactive impurities
  • Batch variation
  • Modified peptides and unnatural amino acids.
• How do you calculate hydrophobicity?