Amino acid analysis: how peptide content is measured

Definitions

• Hydrolysis: cleavage of every peptide bond, classically in 6 M hydrochloric acid at about 110 °C for around 24 hours.
• Derivatisation: chemical tagging of amino acids so they can be detected by UV or fluorescence after separation.
• Isotope dilution: adding known amounts of isotope-labelled amino acids as internal standards for mass spectrometry.

The workflow

• Weigh the sample accurately and hydrolyse it.
• Separate and quantify the released amino acids by chromatography, with derivatisation or by LC-MS.
• Compare the measured amounts with the expected composition from the sequence.
• Use the stable, well-recovered residues to calculate moles of peptide, then mass of peptide as a percentage of powder weight.

Source: Amino acid analysis, Curr Protoc Protein Sci 2009 (PubMed 19937719); Amino acid analysis for peptide quantitation using RPLC-MRM-MS, Anal Bioanal Chem 2023 (PubMed 37468754)

Known sources of error

Acid hydrolysis destroys tryptophan and partly destroys serine and threonine; asparagine and glutamine are converted to aspartic and glutamic acid; cysteine and methionine need special treatment. Bonds between bulky hydrophobic residues hydrolyse slowly. Hydrolysis time is therefore a balance between complete cleavage and loss of labile residues, which is why reports name the residues used for the calculation.

Isotope-dilution mass spectrometry reduces some of these errors and is used to certify protein and peptide calibrants.

Source: The effect of hydrolysis time on amino acid analysis, J AOAC Int 2005 (PubMed 16001867); Quantification of protein calibrants by AAA with isotope dilution MS, Anal Biochem 2011 (PubMed 20807498)

Reading an AAA-based content figure

• Check which residues were used to calculate content.
• Check that the ratio of residues is consistent with the sequence; a large deviation can indicate a wrong or impure material.
• Remember that content and HPLC purity are different numbers (see purity vs net peptide content).
• Make sure the result is for the same lot as the printed lot number on your vial.

At Sable

There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.

Release is at ≥99% HPLC purity with identity confirmed by mass spectrometry.

Frequently asked questions