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How are the structure and purity of a peptide validated?

The purity requirements for synthetic peptides vary depending on their desired us. For some experiments or uses, the purity of the peptide is not critical. However, in other cases, a peptide that is highly pure, free of racemization, deletions, or side chain modifications may be required. Historically, thin layer chromatography (TLC) and analytical reversed-phase high performance liquid chromatography (HPLC) were used to check the purity and if the purification strategy planned has a high probability of success. As an example, if all the peaks in the chromatogram are widely separated when using analytical HPLC, low-pressure reversed-phase HPLC will work as well. However, in high-throughput setting using automated solid-phase peptide synthesis (SPPS), high-pressure preparative HPLC is now commonly used. More modern techniques such as mass spectrometry, e.g. MALDI-TOF-MS and LC-ESI-MS(MS) based methods are now more routinely used.

Techniques of choice for verifying the structure and purity of a synthetic peptide are:

AAA

Amino acid analysis.

Mass Spectrometry

Matrix Assisted Laser Desorption Time Of Flight Mass Spectrometry (MALDI-TOF-MS) and Electrospray Ionization mass spectrometry (ESI-MS).


However, no single technique or procedure for characterization of biomolecule is totally foolproof it is advised to use multiple techniques for the analysis of the final product.

Drawbacks for each method to keep in mind are:

Technique

Drawback

Acid hydrolysis.

 

Standard : 6 N HCl 110 °C, 24 hours.

Hydrolysis in acid is routinely used to cleave peptide bonds for AAA. Some amino acids are destroyed during hydrolysis such as tryptophan and cysteine. Some are only partially recovered, such as serine and threonine. Consecutive hydrophobic sequences are also hard to cleave and may need optimized cleavage conditions.

Also, acid-labile side-chain-protecting groups will not be detected as well in a peptide product.

Enzymatic digestion.

Enzymatic digestions used prior to AAA may overcome some of the problems encountered during acid hydrolysis, but of then enzymatic digestions is incomplete. Furthermore, there is no universal enzyme that will cleave all the needed peptide bonds. For example, if dipeptides are left in the digest, data analysis will be difficult.

Edman chemistry based sequencing.

Often the C-terminal end of the peptide is not determined or observed during Edman based sequencing. If lysine (K) and arginine (R) are not present at the C-terminal end, the last few amino acids tend to wash out and the signal for these amino acids is lost.

Mass Spectrometry.

Not all peptide ionize the same or are volatile in the vacuum of the mass spectrometer. Therefore, the ration of volatilized products may not be representative of the actual peptide present. If this is the case, this type of analysis will not be quantitative.


Many books are available for review covering the techniques for peptide purification and analysis.

Selected references for books

Allen, G., ed. 1989. Laboratory techniques in biochemistry and molecular biology. Sequencing of proteins and peptides. Col. 9 Elsevier, Amsterdam.


Bodansky, M.; Principles of Peptide Synthesis.

Boyer, R. 2012. Biochemistry Laboratory. Modern theory and techniques. Pearson.

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