Amino Acid Analysis (Assay)

Amino Acid Analysis refers to the methodology used to determine the amino acid composition or content of proteins, peptides and other pharmaceutical preparations. Proteins and peptides are macromolecules consisting of covalently bonded amino acid residues organized as a linear polymer. The sequence of the amino acids in a protein or peptide determines the properties of the molecule. Proteins are considered large molecules that commonly exist as folded structures with a specific conformation, while peptides are smaller and may consist of only a few amino acids.

Amino Acid Analysis can be used to quantify protein and peptides, to determine the identity of proteins or peptides based on their amino acid composition, to support protein and peptide structure analysis, to evaluate fragmentation strategies for peptide mapping and to detect atypical amino acids that might be present in a protein or peptide.

It is necessary to hydrolyze a protein/peptide to its individual amino acid constituents before amino acid analysis. Following protein/peptide hydrolysis, the amino acid analysis procedure can be the same as that practiced for free amino acids in other pharmaceutical preparations. The amino acid constituents of the test sample are typically derivatized for analysis.

Analysis of amino acids is required in several areas of research, and it is a fundamental tool in product analysis. The application imposes different requirements on the analytical method because the amino acids play different roles.

• Amino acids are the basic constituents of proteins. Qualitative and quantitative analysis of the amino acid composition of hydrolyzed samples of pure proteins or peptides is used to identify the material and to directly measure its concentration.
• Amino acids are also intermediates in metabolic pathways, often not directly involving proteins. The amino acids are measured as elements of physiological and nutritional studies. This has proven particularly important in monitoring the growth of cells in cultures, as used in the production of biopharmaceuticals.
• Similar considerations lead to the analysis of foods and feeds to ensure that nutritional requirements are met.

The UPLC Amino Acid Analysis has the following features:

• Method robustness minimizes variability
• Better resolution translates into accurate results
• UPLC delivers greater throughput than traditional HPLC
• Protein characterization accelerates for comparison of amino acid composition deduced from the gene sequence for a product or its natural counterpart
• Monitoring cell culture media for critical nutrients allows you to optimize expression efficiencies and to make timely product harvesting decisions

Amino Acid Analysis is an important tool in research and product development in the food, feed, drug and supplement industries.  It is the most accurate way to determine the composition and quantity of protein in a sample.  Amino Acid Analysis is the suitable tool for precise determination of protein quantities; it also provides detailed information regarding the relative amino acid composition and free amino acids. The relative amino acid composition gives a characteristic profile for proteins, which is often sufficient for identification of a protein.  It is often used as decision support for choice of proteases for protein fragmentation.  There are four steps in Amino Acid Analysis:

1. Hydrolysis
2. Derivatization
3. Separation of Derivatized Amino Acids
4. Data Interpretation and Calculations

1. Hydrolysis
A known amount of internal standard is added to the sample. Since norleucine does not naturally occur in proteins, which is stable to acid hydrolysis and can be chromatographically separated from other protein amino acids, it makes an excellent internal standard. The molar amount of internal standard should be approximately equal to that of most of the amino acids in the sample. The sample, containing at least 5 nmoles of each amino acid is then transferred to a hydrolysis tube and dried under vacuum. The tube is placed in a vial containing 6N HCl and a small amount of phenol and the protein is hydrolyzed by the HCl vapors under vacuum. The hydrolysis is carried out for 65 minutes at 150 deg. C. Following hydrolysis, the sample is dissolved in distilled water containing EDTA and approximately 1 nmole of each amino acid is placed on a glass amino acid analyzer sample slide. Hydrolysis can have varying effects on different amino acids.

2. Derivatization
The free amino acids cannot be detected by HPLC unless they have been derivatized. Derivatization is performed automatically on the amino acid analyzer by reacting the free amino acids, under basic conditions, with phenylisothiocyanate (PITC) to produce phenylthiocarbamyl (PTC) amino acid derivatives. This process takes approximately 30 minutes per sample. A standard solution containing a known amount (500 pmol) of 17 common free amino acids is also loaded on a separate amino acid analyzer sample spot and derivatized. This will be used to generate a calibration file that can be used to determine amino acid content of the sample. Following derivatization, a methanol solution containing the PTC-amino acids is transferred to a narrow bore HPLC system for separation. See Table 2 and Table 3 for effects of contaminants on derivatization.

3. HPLC Separation
The PTC-amino acids are separated on a reverse phase C18 silica column and the PTC chromophore is detected at 254 nm. All of the amino acids will elute in approximately 25 minutes. The buffer system used for separation is 50 mM sodium acetate pH 5.45 as buffer A and 70% acetonitrile/32 mM sodium acetate pH 6.1 as buffer B. The program is run using a gradient of buffer A and buffer B with an initial 7% buffer B concentration and ending with a 60% buffer B concentration at the end of the gradient.

4. Data Interpretation and Calculations

Chromatographic peak areas are identified and quantitated using a data analysis system that is attached to the amino acid analyzer system. A calibration file is used that is prepared from the average values of the retention times (in minutes) and areas (in Au) of the amino acids in 10 standard runs. Since a known amount of each amino acid is loaded onto the analyzer, a response factor (Au/pmol) can be calculated. This response factor is used to calculate the amount of amino acid (in pmols) in the sample. The amount of each amino acid in the sample is calculated by dividing the peak area of each (corrected for the differing molar absorptivities of the various amino acids) by the internal standard (norleucine) in the chromatogram and multiplying this by the total amount of internal standard added to the original sample. After the picomole by height of each amino acid has been calculated, the data can be manipulated to yield more useful information.

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