During Amino Acid Analysis, proteins and peptides are hydrolyzed to their component amino acids which are then separated by HPLC, detected by UV or flourometry and quantitated.
When Amino Acid Analysis is used to measure the free amino acids that are not bound up in a protein or peptide but exist freely in a biological tissue like serum, spinal fluid and cells, hydrolysis is not used. Usually, higher molecular weight proteins and peptides are removed from the matrix by a filter. However, the assay is “blind” to the peptides and proteins and only the free amino acids are derivatized and seen in the working part of the chromatogram.
Amino Acid Analysis can be used to answer many biological questions. New technologies have permitted the assay to be scaled down and allow greater sensitivity and the analysis of less abundant samples which increases the usefulness of this assay greatly.
The assay is primarily used as the gold standard in protein and peptide quantification. The Bradford and other colorimetric assays are easy to perform and do not require much instrumentation. But these assays are not accurate and vary from sample to sample. The colorimetric assay can be calibrated, however, to an amino acid analyzed standard if one is working with the same protein(s) repeatedly.
Amino Acid Analysis is a useful tool in synthetic peptide qualification. The mole percent of each residue can be compared to the expected percents from the known sequence.
Some data-based searching can be done from the percent composition of a protein determined by amino acid analysis. But Edman chemistry and Mass Spectrometry are much more selective, have higher confidence limits and therefore are the analyses of choice for protein identification.
Amino Acid Analysis can be used in many creative ways. Just a few of the many past and present projects that utilize this service in our lab are:
- Qualification of MAPS peptides that cannot be easily purified and are not compatible with Mass Spectrometry
- Determination of resin loading
- Determination of drug loading on nanoparticles
- Measuring the rate of enzymatic efficacy in the hydrolysis of di-peptide libraries
- Measuring the changes in culture media amino acids over time
- Measuring the accumulation of marker amino acids (ex. DAPA in bacterial cell walls) over time
- Monitoring organic matter in drinking water related to the production of trihalmethanes from chlorination
- Quantitation and qualification of recombinantly produced proteins
- Measuring changes in Glutamic Acid in CNS during brain insult
- Measuring changes in percent lysine in grains at different stages of growth