4 things to remember about Amino Acid Analysis of proteins & peptides

September 23 2016, by Ejvind Mørtz

Ejvind Mørtz, co-founder and COO

Why perform Amino Acid Analysis?

Amino acid analysis of proteins is an excellent method to determine the absolute amounts of individual amino acids in a sample. The technique is applicable for samples that contain free amino acids. However, it is also helpful for peptides and protein samples after hydrolysis into amino acids [1, 2].

With AA analysis you can determine the relative composition of amino acids in a protein. Determining the absolute amount of a protein or peptide is also recommended if the amino acid sequence is known. In addition, it is a crucial part of the extinction coefficient determination of a protein or antibody of interest. Finally, the analysis is ideal for the purity estimation of a purified protein [3].

We recommend that you determine the protein concentration of your sample before any host cell protein analysis and other quantitative analyses. The results aid in calculating the appropriate sample volume for protein digestion and preparation for mass spectrometry-based workflows, thus resulting in more accurate and valuable data.


When using amino acid analysis, you should note that:

  1. Serine and threonine degrade slightly during acid hydrolysis, and recoveries are sometimes up to 10% lower than expected.
  2. Methionine can oxidize during hydrolysis; however, less than 10% is usually oxidized.
  3. Valine and isoleucine bonds (Val-Val, Ile-Val, Val-Ile, Ile-Ile) are challenging to hydrolyze, and recoveries are often 5-15% lower than expected.
  4. Glycine content is often higher than expected because it is a frequent contaminant from many buffers. Determining a compensation factor to correct for differences in ninhydrin reactivity uses an analysis of known amounts of AA standards. [1-3].

Hopefully, you now have a good idea of what to observe when performing amino acid analysis. If you need more information, you may want to visit this page to read more about Amino Acid quantification.


Related blog posts on amino acid analysis:

I highly recommend that you spend some time familiarizing yourself with the information in these blog posts:

Important reasons why you need to determine the concentration of your protein standards accurately

How to determine the Molar Extinction Coefficient of protein in 3 small steps


[1]          Rutherfurd et al.: “Amino acid analysis, “Current Protocols in Protein Science, 2009

[2]          Rutherfurd et al.: “Quantitative amino acid analysis, “Current Protocols in Protein Science, 2011

[3]          Noble et al.: “Quantification of protein, “Methods in Enzymology, 2009

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