Peptide Synthesis: Handling and Storage of Synthetic Peptides

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Frequently Asked Questions (FAQ)

References using synthetic peptides and antibodies from LifeTein. Full Publication List of 2017.

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What is peptide purity?

Peptide purity is the amount of the target peptide as determined by HPLC at 214 nm, where the peptide bond absorbs. Water and residual salts are not detected by a UV spectrophotometer. Other impurities that can be found in the content include deletion sequences (shorter peptides lacking one or more amino acids of the target sequence), truncated sequences (generated by capping steps to avoid the formation of deletion peptides), and incompletely deprotected sequences (generated during the synthesis or the final cleavage process).

Peptide purity does not include any water or salts in the sample. TFA results from HPLC purification. The free N-terminus and other side chains such as Arg, Lys, and His form trifluoroacetate, and this allow small amounts of TFA to contaminate the peptides. Peptides are usually delivered as trifluoroacetate containing residual water. Even in lyophilized peptides, varying amounts of noncovalently bound water still exist.

What are other substances (impurities) in the peptides?

Impurities

Non-Purified Peptides

Purified Peptides (HPLC)

Deletion sequences1

Truncation sequences2

Incompletely deprotected sequences3

Sequences modified during cleavage4

DTT (dithiothreitol)

TFA (trifluoroacetic acid)

Acetic acid

Peptides that have undergone side reactions such as proline isomerization or isoaspartimide formation, etc.

The impurities in non-purified peptides are both peptides and non-peptides, the impurities in purified peptides are mostly peptides with modified sequences, except for TFA salt.

  1. Shorter peptides lacking one or more amino acids of the target sequence
  2. Generated by capping steps to avoid the formation of deletion peptides
  3. Generated during the synthesis or the final cleavage process
  4. Reattachment of protecting groups at other locations on the peptide