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Our analysis methods

Tryptophan and cysteine are generally found in proteins and may also be incorporated in peptides. For the analysis of these specific amino acids in samples, AltaBioscience uses different methods that enable us to accurately quantify both Cys and Trp.

Separate individual analyses must be performed for the accurate determination of tryptophan, cysteine and cystine amino acids. Without pre-treatment or special analysis, the recovery of these amino acids is affected or lost altogether.

During standard analysis, Tryptophan is destroyed using conventional acid hydrolysis but is stable to alkaline hydrolysis with barium hydroxide. We are able to provide a separate alkaline system that will ensure the tryptophan is not destroyed and can be accurately quantified.

Cysteine is also unstable to conventional acid hydrolysis. It is usually observed as cystine and its recovery is variable using standard hydrolysis conditions. However, oxidation to cysteic acid using performic acid prior to acid hydrolysis gives very good recovery and accurate quantification of Cys. As the recovery of other amino acids in the sample may be affected by this pre-treatment, we must run this type of analysis in addition to our standard hydrolysis run.

To summarise, both these methods adversely affect recovery of the other amino acids, therefore in order to quantify all 20 amino acids, three completely separate analyses are required: acid hydrolysis, alkaline hydrolysis and acid hydrolysis with performic acid oxidation.

We can, if the peptide sequence is known, estimate the values for Cys and Trp but for accurate determination, we need to perform separate analyses as detailed above.

Tryptophan, Cysteine and Cystine structures

Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a nonpolar aromatic amino acid. It is essential in humans, meaning that the body cannot synthesise it and it must be obtained from the diet, since it is critical in a number of metabolic functions. Therefore, if analysis of the essential amino acids is required for a sample, a total amino acid analysis and separate analysis of Tryptophan must be carried out in addition to total amino acid analysis.

Cysteine is a non-essential amino acid in humans. It is important for protein synthesis, including synthesis of beta-keratin, the main protein in nails, skin, and hair, and also collagen production, as well as skin elasticity and texture. Cysteine is a unique amino acid as it has a very reactive sulfhydryl group at its side chain, which is capable of forming disulphide bridges. Disulphide bridges play a critical stabilising role in many protein structures by forming cross-links between different regions of polypeptide chains. If a peptide for analysis contains a disulphide bridge, it must be treated with DTT prior to separate analysis of Cysteine. Please contact one of our technical experts for further information.

Cystine is the oxidised dimer form of cysteine. Two molecules of cysteine are joined together by a disulphide bridge to form cystine.

Read more about our amino acid analysis services.

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