Amino acid analysis for protein quantification

It is advisable to determine the protein concentration of your sample by amino acid analysis before conducting other quantitative analyses, such as residual HCP quantification or peptide and intact mass analyses. The data ensures the appropriate sample volume and protein amount calculations for protein digestion and mass spectrometry-based workflow preparation. If the protein concentration has been incorrectly measured, the analysis may fail or the results may be less accurate.

The amino acid analysis is the golden standard for accurately quantifying the specific amount of purified proteins and peptides and also for determining the overall protein concentration in complex protein samples. The analysis provides quantitative data for all amino acids except tryptophan and cysteine, which degrade into free amino acids during hydrolysis. The samples can even be run in triplicate for highly accurate quantification.

Traditional protein quantification assays, such as Bradford, Lowry, or BCA, depend on an external reference standard and calibration curve and are prone to interference from chemicals or the presence of other proteins. Furthermore, your purified protein may have a different UV absorption coefficient than the employed calibration standard. Such cases will result in an incorrect concentration determination of the protein standard, which may significantly impact your assay and downstream process development.

Triplicate amino acid analysis (AAA) is the gold standard for protein concentration determination. Generally, it ensures the most accurate quantification of your protein standard, even when the concentration is unknown. The analysis is based on acidic hydrolysis followed by Ion-Exchange HPLC detection and quantifying individual amino acids. The analysis works even with presence of salt and certain detergents.

Knowing the molar extinction coefficient of your purified protein or antibody sample is necessary for accurate protein quantification by UV absorbance and is also required by the ICH Q6B guidelines. The extinction coefficient enables the determination of protein concentrations in laboratory samples using spectrophotometry.

The analysis is a three-step procedure of measuring the exact protein concentration, determining its UV absorbance, and calculating the extinction coefficient using the Beer-Lambert Law. The protein or antibody should be dissolved in the same buffer for subsequent experiments since the molar extinction coefficient depends on the protein's buffer composition and three-dimensional structure.

The method usually requires access to an amino acid analyzer and a spectrophotometer. Alphalyse uses an advanced SoloVPE system with variable-path length measurement, which can identify the linear range of absorbance and allows the calculation of the sample concentration and extinction coefficient using a slope spectroscopy equation derived from the Beer–Lambert Law.