What Are the Key Steps in Protein Amino Acid Analysis, and Why Do Reports Typically Show 17 Amino Acids?
The key steps in determining the amino acid composition of proteins are as follows:
1. Protein Hydrolysis
The protein sample is hydrolyzed using strong acid, typically 6M hydrochloric acid, under high-temperature conditions (e.g., 110°C for 24 hours) to break it down into free amino acids.
2. Purification of Hydrolysate
The hydrolyzed sample may contain non-amino acid byproducts, requiring purification through techniques such as ion exchange chromatography.
3. Derivatization
Some amino acids require chemical derivatization to improve their detectability and quantification in analytical systems.
4. Separation and Quantification
High-performance liquid chromatography (HPLC) is commonly used to separate and quantify individual amino acids. The peak area of each amino acid is proportional to its concentration, allowing precise quantification.
5. Data Interpretation
The amino acid content is determined by comparing the measured peak areas to calibration curves or reference standards.
Regarding why typical analysis reports include 17 amino acids instead of all 20, this is due to the instability of certain amino acids under acid hydrolysis. Specifically:
Why Are Only 17 Amino Acids Typically Reported?
Although proteins are composed of 20 standard amino acids, conventional amino acid analysis usually detects only 17. This is because three amino acids-tryptophan, cysteine (or cystine), and lysine-are prone to degradation or chemical transformation during acid hydrolysis:
1. Tryptophan readily degrades under acidic conditions.
2. Cysteine and cystine undergo oxidation and are converted into other derivatives.
3. Lysine may undergo side reactions that alter its detectability.
Due to these limitations, standard analytical methods primarily report the remaining 17 amino acids.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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