Protein Facility

NOTE: This is for informational purposes only, we do not offer this as a service.
Please contact the University of Missouri Agricultural Experiment Station Chemical Laboratories for amino acid analysis service.

Amino acid analysis is a process to determine the quantities of each individual amino acid in a protein. 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 [Top]
  A known amount of internal standard (norleucine) is added to the sample. Since norleucine does not naturally occur in proteins, 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 (i.e. 10 ug of protein) is then transferred to a hydrolysis tube and dried under vacuum. The tube is placed in a vial containing 6 N 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 (to chelate metal ions) 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 (see Table 1).
  
  
• 2. Derivatization [Top]
  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 (see Figure 1 and Figure 2). 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 [Top]
  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 (see Figure 3).
  
  
• 4. Data interpretation and calculations [Top]
  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.
  
  • Mole percent represents the amount of each amino acid present as a percentage of the total amino acids recovered in the sample. Mole percent can be useful for samples in which there is no known composition or molecular weight, non-specific molecular weights, or the sample contains mixtures of proteins, free amino acids and other components.

    [pmol of individual amino acid] / [total pmol of all amino acids in the sample] X 100 = mole percent of each amino acid

  • Composition by molecular weight can be used when the molecular weight of the sample is known and the amino acid composition is desired.

    [pmol of amino acid] X [residue molecular weight of amino acid] = picogram of amino acid

    sigma[picogram of all amino acids] / [molecular weight of sample] =[pmol of sample recovered]

    [pmol of amino acid] / [pmol of sample recovered] = # of residues of amino acid per molecule of sample

  • Composition by residue is used when the amino acid composition is required and the number of times a particular amino acid residue occurs in the sample is known.

    [pmol of selected amino acid] / [known # of residues of selected amino acid/sample molecule] = pmol of sample recovered

    [pmol of amino acid] / [pmol of sample recovered] = # of residues of amino acid per molecule of sample

  • Minimum molecular weight is performed if neither the composition or the molecular weight of a pure protein or peptide is known. 1. The amino acid present in the smallest amount is determined and assigned a value of one. 2. The amount of this amino acid is then divided into the amount of all the other individual amino acids. The estimated composition based upon the previous calculations is then used to calculate the minimum molecular weight of the sample. The minimum molecular weight of the sample is merely an integer of the actual weight.

Table 1 Acid hydrolysis effects on various amino acids

Table 2 Effects of common contaminants on derivatization yield

Table 3 Effects of trace contaminants on amino acid derivatization