Why you need accurate concentration determination of protein standards

Question:

How do I quantify my protein standard?

How do I quantify my protein standard? Is it true that it if it is done incorrectly it can affect the determination of my protein concentration?

Answer:

Have you ever questioned the quantitative data you get from Bradford, Lowry or BCA protein quantification assays? These traditional methods are very fast and can be performed in most laboratories. They are based on UV absorption (A280) of the protein relative to a standard, typically BSA, of known concentration [1, 2].

• Unfortunately, these assays are prone to interference from chemicals and the presence of other proteins.
• Furthermore, your purified protein may have a different UV absorption coefficient than the BSA calibration standard.

Therefore the concentration determination of the protein standard can be wrong! The result is that all your experiments based upon the amount in the protein reference standard could also be wrong! Imagine the impact this will have on your assay or your downstream process development [1, 2].

This is why you should use triplicate amino acid analysis (AAA) for accurate quantitation of your protein standard [3].

Amino acid analysis – the gold standard for protein concentration determination

There are several good reasons why you should use triplicate AAA [2] when you need quantitative data you can trust – for instance to analyze your protein standard solutions:

1. It is a very robust analysis. It can be performed in the presence of salt and detergents, as it is based on ion-exchange chromatography. The only contaminant that can obstruct the analysis is a chemical containing primary amines. And even then, it might still be possible to do an accurate quantification if the calculations can be based on amino acids not affected by the contaminant.
2. You can compare the results with the sequence. This is a very important feature of the analysis. By comparing the quantitative data for each amino acid residue with the amino acid sequence of the protein you will get information about the purity of your sample. If the data does not fit within a few residues, your sample is most likely contaminated by other proteins.
3. It is linear over a broad concentration range. The analysis can measure from 100-10.000 pmol with high linearity. Therefore, it is suitable for samples with an unknown protein concentration.
4. Your data can be qualified, using internal and external reference standards. You obtain the most accurate result by analyzing each sample in triplicate. This means that each sample is analyzed as 3 individual samples all the way from hydrolysis through data calculation. To determine the precision and the accuracy of the test you use protein standard solutions in each batch analysis. Adding an internal standard amino acid (e.g. Sarcosine or Norleucine) to each sample makes it possible to correct changes in reagent stability and HPLC flow rate [1-3].

How do we approach protein concentration determination at Alphalyse?

At Alphalyse, we quantify sixteen amino acids. These are the normal 20 amino acids with the exception of tryptophan and cysteine, which are degraded by the hydrolysis process. We thus determine Asparagine as aspartic acid and glutamine as glutamic acid. I always suggest running each sample in triplicate and we also include two BSA samples (NIST standard) to determine the precision and the accuracy for each batch analysis.

Figure: HPLC separation of amino acids for identification and quantification

In order to calculate the data, you first take the measured AA amount in pmol divided by the number of each specific amino acid in the sequence providing a pmol protein. Then you calculate the average of these numbers in pmol using the amount of protein in µg and the molecular weight of the protein.

Finally, we perform the calculations using a best linear fit based on detected amino acids. We modify each experiment, in order not to include amino acids with greater than 5 per cent variation.

Table: Calculating the accurate amount of protein

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References:

[1]          Rutherfurd et al: “Amino acid analysis“, Current Protocols in Protein Science, 2009

[2]          Rutherfurd et al: “Quantitative amino acid analysis“, Current Protocols in Protein Science, 2011

[3]          Noble et al: “Quantification of protein“, Methods in Enzymology, 2009