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Purity
Purity of Synthetic Peptides
Because of the nature of chemical synthesis, all synthetic peptides contain side
products that result from incomplete coupling of the next amino acid (e.g., n-1,
n-2... deletions), incomplete deprotection (e.g., incomplete removal of trityl
protecting groups from reactive side chains), and from side reactions (e.g.,
oxidation of methionine) that may occur during synthesis, cleavage, or purification.
Obviously, the relative concentrations of these side products likely will be significantly
higher in the crude as compared to in the purified peptide. It must be kept in mind that
these side products may be either more or less immunogenic or "active" than the
desired product. Hence, care should be exercised especially when interpreting the results
of studies carried out with crude synthetic peptides. While analytical reverse phase
HPLC provides one of the best means of quickly assessing the complexity of synthetic
peptides there are several caveats that must be kept in mind:
- Symmetrical absorbance peaks may contain multiple components that failed to resolve.
- Some peptides (e.g., hydrophilic peptides less than a few residues in length) may
not bind to the C18 column and some peptides (e.g., hydrophobic peptides
corresponding to membrane spanning regions) may not elute or may only partially elute.
- Since peptide elution is monitored via absorbance at 210 nm, the relative abundance of
components that contain species that absorb strongly at this wavelength (e.g., incompletely
deprotected peptides that contain the Trityl group) will be over-estimated.
Although it is probable that the major absorbance peak on the RP-HPLC chromatogram
corresponds with the major ion peak detected by MALDI-MS, it is possible that this often
made assumption is not valid for some peptides. That is, while individual fractions from
the RP-HPLC purification of synthetic peptides are subjected to both analytical
HPLC and MALDI-MS, this important correlation must be assumed in the case of crude
peptides where the MALDI-MS is not carried out on an HPLC purified fraction.
Similarly, since minor changes in structure may substantially influence MALDI-MS response,
the relative intensities of different ions may not be linearly related to their
concentrations.
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