Why is accurate protein quantification important?
Quantification is an essential part of protein analysis and quality control. It means that you can determine protein yield and that way measure the success of your experiments and compare them to others. Accurate quantification is especially important when analysing potency and kinetics under different conditions, when making comparisons between mutant and wild type specimens, or when developing and testing new drugs, as even small changes in protein concentration can dramatically alter results.
As such, much research has been dedicated to finding the best methods, both in terms of accuracy and ease of use. This has resulted in an array of available assays, each with its own strengths and limitations.
UV-Vis spectroscopy (A280)
Most proteins contain tryptophan or tyrosine amino acid residues and cystine disulfides, which absorb UV light at 280 nm. This means that the protein concentration can be obtained using UV-Vis spectroscopy, by applying the Beer-Lambert law, A = εcl , where A is absorbance at 280 nm, ε is the extinction coefficient, c the concentration and l the path length.
Its simplicity is both its main strength and main weakness: it’s easy, but it’s really only suited to straight-forward cases. For example, the extinction coefficient – a measure of how strongly a substance absorbs light and in the equation above – must be known, otherwise the Beer-Lambert law is not much use. As the vast majority of the absorbance signal comes from tryptophan, any proteins or peptides that are lacking in tryptophan are difficult to measure accurately using UV-Vis. If you want to measure a mixture of proteins, it becomes even trickier.
It also struggles with contamination from nucleic acids and cell fragments, though certain modern UV-Vis spectrophotometers can somewhat adjust the results to counteract some of the error.
The Bradford assay is based on the complexing of the dye Coomassie Brilliant Blue G-250 (CBB) to proteins, which shifts the colour of the dye from red to blue. If there is no protein present, no complex is formed and the dye remains red. The absorbance at 595 nm corresponds to the amount of blue CBB, and this in turn gives the concentration of protein present.
To convert the absorbance to concentration, a standard curve is used. Bradford assays are subject to great protein-to-protein variation, meaning that the absorbance obtained for the same mass of different proteins will vary. That’s why it’s preferable to find a standard that reacts similarly to the sample protein, though in practice this is often unfeasible and a much less specific standard has to be used.
This method is susceptible to detergent contamination, usually from lysing, because certain detergents will preferentially bind the proteins and not allow CBB to complex. To complicate the issue further, the same detergent might in higher concentrations bind CBB to produce its blue form. In both cases, the concentration will be off.
The whole technique is clearly very dependent on the relationship between the concentration and the absorbance. Only in a narrow concentration range is this relationship linear, significantly complicating quantification outside this range. To avoid this, proteins must be diluted, sometimes significantly, to fit into the linear concentration range.
Other colourimetric assays that don’t suffer as much from detergent contaminations or have larger linear ranges have been developed, but these often suffer from higher protein-to-protein variation or vice versa. The choice of any such assay is therefore a trade-off.
Amino acid analysis (AAA)
In this technique, the protein is hydrolysed overnight such that the amino acids are released. They can then be separated by chromatography and detected by spectroscopy, generally after a modification which will render them detectable. A mixture of amino acids at several different concentrations is used to create the standard curve from which concentration will be determined.
Similar to colourimetric assays, buffer components such as detergents or salts will cause errors. However, the protein hydrolysis is the most sensitive part of the analysis. It must be carried out with care because acid hydrolysis in particular (which is the most common method of amino acid analysis), can result in the complete or partial destruction of certain amino acids. As such, the hydrolysis technique must be chosen carefully to suit the sample to avoid large errors.
Amino acid analysis is considered the “gold standard” in protein quantification, but requires specialised equipment and expertise and so is concomitantly expensive – per sample cost can range from £12 per run (~$17) if available through a departmental service up to £100 per run (~$140) if using an commercial provider.
Because it does not require that proteins contain tryptophan and/or tyrosine residues like UV/Vis, it is suitable for quantifying peptides without these amino acids.
Gel electrophoresis can also be used to determine protein concentration. A gel of the unknown protein and different concentrations of the standard, usually bovine serum albumin (BSA), is run, and the intensity of each standard band is then plotted against mass. The unknown protein concentration can then be determined by comparison to the standard curve.
However, the band intensity depends not only on mass but differs from protein to protein, which makes it problematic to infer concentration based on an unspecific standard like BSA.
Additionally, it’s important to ensure that bands are not overloaded, do not saturate the detector, and to stay consistent in the way that the band intensity is estimated, which can be done with image analysis software, like ImageJ, or a densitometer, because this too can introduce inaccuracies.
Protein quantification on the Fluidity One
Fluidity One uses an amine reactive fluorogenic dye to determine protein quantification. 5-10 µL of protein sample is pipetted onto a disposable chip and inserted into the instrument. Quantification takes approximately 8 minutes.
Upon reaction with the protein’s amine groups, the dye becomes fluorescent and the concentration is determined from the fluorescence intensity. The dye will bind lysine residues and also the unprotected N-terminus.
The unbound dye does not fluoresce, ensuring a low background and high sensitivity. Proteins can be quantified down to a concentration of 10 ng/µL for most proteins. This will be somewhat dependent on the composition of the protein being studied, and reflects sensitivity and saturation of the underlying detection chemistry.
As with amino acid analysis, there is no requirement for tryptophan or tyrosine residues, and so the approach can be used to quantify any protein or peptide. However, the amine reactive nature of the dye means that the sample must be free of any other primary amines, such as Tris buffer or free amino acids.
Protein quantification - the right tool for the job...
It is apparent from the various strengths and limitations of each protein quantification method that there will be occasions where each is the right tool for the job (Table 1).
|UV-Vis (NanoDrop Cuvette)||Bradford (NanoDrop)||Amino acid analysis||Gel electrophoresis||Fluidity One|
|Method of detection||Absorbance of tryptophan and tyrosine residues||Absorbance of Coomassie Brilliant Blue G-250 complex||Direct detection of modified amino acids||Gel band intensity||Fluorescence of amine reactive dye|
10-2000 ng/µL (BSA)
Molarity: 150 nM – 30 µM
15-100 ng/µL (BSA)
Molarity: 225 nM – 1.5 µM
0.006-0.7 ng/µL (BSA)
Molarity: 0.1- 10 nM1
50-200 ng/µL (BSA)
Molarity: 0.75 – 3 µM
5-500 ng/µL (BSA)
Molarity: 75 nM – 7.5 µM
Requires protein or peptide to contain tyrosine/tryptophan
Extinction coefficient must be known
Struggles with contamination
Narrow linear range
High protein-to-protein variation
Susceptible to detergent contamination
Slow and expensive
Free amines may contaminate results
High protein-to-protein variation
Requires time-consuming image analysis or densitometry
|Sample must not contain other free amines|
The Fluidity One is optimum in instances where a result is required quickly with low concentrations and high accuracy, for example prior to kinetics analysis. The advantage of the Fluidity one is that in addition to concentration, a determination of the protein samples average hydrodynamic radius is also obtained, providing insights into it constituents' dispersity, aggregation state or conformation - and thereby the quality and suitability of that prep for downstream applications.