Total Amino Acid Analysis in mixtures
Amino Acid Analysis (AAA) – Explained
Amino acid analysis remains the gold standard for determining protein concentrations in biological specimens. APAF has provided AAA for Australia’s academic and industry researchers since 1995. AAA can be carried out on free amino acids or by total protein hydrolysis.
Total Amino Acid Analysis
Amino acid analysis at APAF is performed using liquid hydrolysis in 6M HCl followed by quantification using a pre-column derivatisation reversed phase (RP) HPLC procedure. The procedure employed is based on the Waters AccQTag amino acid analysis methodology but has been improved to run on an Ultra Performance liquid chromatography (UPLC) system. APAF has three dedicated UPLCs for AAA with a weekly capacity for analysis of 100 samples.
At APAF, our standard analysis quantitates sixteen standard amino acids. Tryptophan and cysteine are degraded during hydrolysis while aspartic acid and glutamic acid are reported as the sum of asparagine and aspartic acid and glutamine and glutamic acid, respectively. We have alternative assays for tryptophan and cysteine and analysis of other non-standard amino acids are available on request. We run each sample in duplicate and include BSA to confirm the accuracy for each batch analysis.
APAF has refined AAA methodologies for a broad range of sample types including:
- Proteins in complex mixtures (food, raw materials etc)
- Biopharmaceutical products
- Pure proteins
- Physiological fluids (see our previous newsletter on Free Amino Acids
More recently free amino acids analysis has been used in nutritional studies, to identify cancer markers and other areas looking for biomarkers
Stepto, N K; Shipperd, B B; Hyman, G; McInerney, B; Pyne, D B. Effects of high-dose large neutral amino acid supplementation on exercise, motor skill, and mental performance in Australian Rules Football players. Appl. Physiol. Nutr. Metab. 2011 Oct; 36(5): 671-81.
Boeuf, P; Aitken,E H; Chandrasiri, U; Chua, C L L; McInerney, B; McQuade, L; Duffy, M; Molyneux, M; Brown, G; Glazier, J; Rogerson, S J. Plasmodium falciparum malaria elicits inflammatory responses that dysregulate placental amino acid transport. PLoS Pathog 2013 Feb; 9(2): e1003153.
Jared R Mayers et el. Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development. nature medicine VOLUME 20 | NUMBER 10 | OCTOBER 2014
Amino Acid Profile Analysis – Results - Shows results for the 16 amino acids
Sample ID: xyz
Name | Amino Acid (-H2O)* (mg/g) | Amino Acid** (mg/g) | Mole% |
Histidine | 1.32 | 1.49 | 2.2 |
Serine | 1.98 | 2.39 | 5.3 |
Arginine | 2.94 | 3.28 | 4.4 |
Glycine | 3.90 | 5.14 | 15.9 |
Aspartic acid | 4.16 | 4.81 | 8.4 |
Glutamic acid | 6.70 | 7.63 | 12.1 |
Threonine | 2.05 | 2.42 | 4.7 |
Alanine | 2.83 | 3.55 | 9.3 |
Proline | 2.78 | 3.29 | 6.7 |
Lysine | 3.26 | 3.72 | 5.9 |
Tyrosine | 1.56 | 1.73 | 2.2 |
Methionine | 1.07 | 1.22 | 1.9 |
Valine | 2.52 | 2.97 | 5.9 |
Isoleucine | 1.83 | 2.12 | 3.8 |
Leucine | 3.83 | 4.44 | 7.9 |
Phenylalanine | 2.18 | 2.45 | 3.4 |
Total | 44.91 | 52.65 | 100.0 |
* Calculation based on amino acid residue mass in protein (molecular weight minus H2O).
** Calculation based on free amino acid molecular weight.
Auto-scaled Chromatograph