WHITE PAPER

SUMMARY

Protein assays are essential techniques in many laboratory workflows, most notably in life science research. Because of the complexity of biological systems, different assays have been developed that differ in their sensitivity and detection range.

Protein assays can be grouped according to their principle of analysis. Colorimetric assays are used to determine protein quantity based on the intensity of color formation when certain amino acids in a sample react with a reagent. Fluorescence-based assays quantitates proteins based on the intensity of fluorescence when certain dyes react with specific types of amino acids. Spectroscopic assays detect proteins based on their absorption or vibrational properties when exposed to either ultraviolet or infrared radiation. Amino acid analysis is considered the gold standard in protein quantitation. Mass spectrometry is the latest and possibly the most sensitive method that can detect nanogram amounts of proteins.

The choice of protein assay depends on factors such as ease of use, cost, sensitivity, and reactivity. It is important to know which assay to use as this could affect the overall success of any protein-related laboratory endeavor.

Introduction

Precise determination of protein concentration is a necessary step in all studies involving proteins. That is why protein assays have become intrinsic tools in many areas of life science research such as molecular biology, cell biology, and biomedicine. A wide variety of methods are now available to quantitate total protein or single protein content of a sample. The accuracy of the methods used can greatly impact calculations in other processes and could amplify errors further down the research trajectory.
Although there are many protein assays to choose from, care should be taken in choosing the most suitable assay for a specific application. Each assay has its own advantages and limitations. In many cases, it may be necessary to use more than one type of protein assay as there is no single protein assay that will yield the most accurate results.

Importance of Protein Assays for Biopharmaceutical Applications

Protein characterization is one of the most important steps in the development of biopharmaceuticals from the initial stages of conceptualization to final product manufacturing. Most biopharmaceuticals are highly complex compounds that require high purity and undergo both upstream and downstream processes. Accurate monitoring of the concentration of biopharmaceutical proteins is therefore essential to determine the purification yield at each step of the manufacturing process. This in turn provides important information on the capacity and efficiency of the production run.

The main objective of protein characterization of biopharmaceuticals is to decrease the time it takes to get a product to market. Thus, quantification of biopharmaceutical proteins is a crucial step to maintain reliability of whole processes for the production and confidence of the final products.

Several studies have reported that even the most common drugs can interfere with protein assays. For instance, aspirin was reported to interfere with the biuret and Lowry assays, whereas penicillin and ascorbic acid interfere with the Lowry and BCA assays. In this regard, choosing the most appropriate protein assay technique is crucial to the success of biopharmaceuticals production.

Protein Assays

Colorimetric assays
Colorimetric assays are the most commonly used assays for quantifying protein concentration. The amino acid composition of a particular sample affects the accuracy of these assays. As the name implies, colorimetric assays produce a colored solution in the visible spectrum in response to the reaction of amino acids in the sample with a reagent. The intensity of the color formed is measured by absorbance photometry. Normally, standard solutions of bovine serum albumin (BSA) are used to produce a calibration curve of absorbance versus mass concentration. The concentration of the analyte-proteins can be calculated on the assumption that they react in the same manner as the BSA standard.

Biuret Assay
The Biuret Assay is one of the simplest and most common protein assay methods. The assay is essentially a test for compounds having a peptide bond. In an alkaline solution containing sodium potassium tartrate, compounds containing two or more peptide bonds form a complex with the cupric ions (Cu2+) in the biuret reagent when heated. The reaction results in a light blue to purple complex that is measured by absorbance photometry at 540 nm. The intensity of the color is directly proportional to protein concentration.

The advantages of the Biuret Assay are its quick turn-around time with relatively few interfering substances. However, it has low sensitivity and can give incorrect color development in buffer solutions containing ammonium sulfate, which generates different colored complexes within the sample.

Bradford Assay
This assay encompasses various preparations using the dye Coomassie Brilliant Blue G-250 for quantitating protein. The basic mechanism involves the binding of the dye to arginine, histidine, phenylalanine, tryptophan, and tyrosine residues. Upon binding protein, there is a metachromatic shift from 465 to 595 nm resulting from the stabilization of the anionic blue form of the dye. Because majority of the observed signal is from reaction with arginine residues, there is a wide protein-to-protein variation when using the Bradford Assay.

The advantages of the Bradford Assay include its ease of use, sensitivity, and low cost of reagents. It can also be adapted to microplate-based assays for high-throughput protein concentration determination. However, the assay is sensitive to various interfering reagents, such as ionic and nonionic detergents and glycosylated proteins. It has also been shown that there are significant variations in the results between different commercial suppliers of Bradford preparations. This is especially true for low-molecular-weight proteins. Therefore, it is best to stick to the same supplier when performing this assay.

Lowry Assay
The Lowry Assay is a two-step procedure: the initial biuret reaction followed by the enhancement stage. The biuret reaction involves the reduction of copper (Cu2+ to Cu+) by the proteins in an alkaline solution. The enhancement stage involves the reduction of the Folin–Ciocalteu reagent (phosphomolybdate and phosphotungstate) that produces a characteristic blue reaction with absorbance maxima at 750 nm. This color development is largely due to the reduced copper-amide bond complex and residues of tryptophan and tyrosine, and to a limited extent cysteine, cystine, and histidine residues.

The Lowry Assay has undergone several modifications to increase its dynamic range, reduce its sensitivity to interfering substances, and increase the speed and resulting stability of the color formation. But this assay has been largely replaced by the more sensitive BCA Assay. The assay’s sensitivity to interfering substances makes it difficult to generate a linear response. Detergents, potassium ions, lipids, and sodium phosphate can also cause precipitate formation during the reaction.

Bicinchoninic acid (BCA)
Similar to the Lowry Assay, color formation in the BCA assay is the result of two reactions. Initially, cupric ions (Cu+2) are reduced to cuprous ions (Cu+) by the protein under alkaline conditions via the biuret reaction. Then, a complex between Cu+ and two molecules of bicinchoninic acid forms, which produces the characteristic purple color. The intensity of the color formed is measured by absorbance photometry at 562 nm. The BCA reaction is temperature dependent.

Different functional groups may exhibit a different reactivity at elevated temperatures, which results in less protein variability. For instance, at 60℃, a higher reactivity of tryptophan, tyrosine, and peptide bonds results in more color formation compared to the same reaction at 37℃.
The BCA assay can be adapted to a microplate-based assay to improve its sensitivity. This assay is sensitive to copper chelators such as EDTA or reagents that can also reduce Cu2+ such as DTT.

Fluorescence-based assays
Various fluorescent probes or dyes can be used to quantitate amino acid mixtures. The assays rely on the detection of the fluorescence emitted by dyes that react with or bind to amino acids in the protein of interest.

Amine derivatization
Amine-labeling derivatization using various fluorescent dyes is a common method to quantitate amino acid mixtures in quantitative amino acid analysis. This technique can also be used to quantitate proteins containing either lysine or a free N-terminus. Upon reaction with amines, the dyes exhibit a large increase in fluorescence that will generate a linear response with increasing protein concentration.

Ophthalaldehyde (OPA), fluorescamine, and 3-(4-carboxybenzoyl)quinoline-2- carboxyaldehyde (CBCQCTM) are the three dyes that have been used to quantitate proteins and amino acids. Fluorescamine reacts directly with the amine functional group, whereas the other two require the addition of a thiol (2-mercaptoethanol) or cyanide (for CBCQCTM).

Amine derivatization offers improved sensitivity and dynamic range when compared with colorimetric and absorbance-based assays. But use of this technique is limited because of the variation in the number of lysine residues in proteins, which results in a large protein-to-protein variability. Buffers containing glycine and amine, ammonium ions, and thiols, which are common in many buffer systems, interfere with the assay, thus, limiting its application. The pH of the reaction also affects the reproducibility of this assay.

Detergent-based fluorescent detection
Fluorescent probes with significantly improved quantum yields when bound at the detergent–protein interface have been used with some success to quantitate proteins in both gels and solutions. The NanoOrangeTM and Quant-iTTM are two commercially available preparations that use this technique.

Both assays are sensitive to detergents and high salt concentrations. Furthermore, the NanoOrangeTM requires samples to be heated to 90℃ to denature the proteins, which is supposed to reduce protein-to- protein variability. On the other hand, the Quant-iTTM assay shows better sensitivity and dynamic range compared to dye-based assays. It also has relatively low protein- to-protein variability.

Spectroscopic assays
Spectroscopic methods are standard techniques for measuring protein concentration in solutions. Protein characterization is performed based on either the absorption or vibrational properties of aromatic amino acids or peptide bonds in a protein, which provides information to determine protein concentration. The two most common spectroscopic methods are ultraviolet (UV) spectroscopy and infrared (IR) spectroscopy.

UV–Visible radiation
The concentration of proteins can be determined by measuring the absorbance of ultraviolet–visible radiation. For biopharmaceutical proteins, concentration is determined based on their absorbance at 280 nm (A280) using a UV–Vis spectrophotometer. The basis for determining the concentration of a biopharmaceutical protein in solution using the A280 value comes from Beer– Lambert’s Law.

This method is both simple and direct with no incubation time or dye addition required. However, it is necessary to have an accurate value of the molar absorption coefficient, ε for using A280 value. Because proteins vary in the number of aromatic amino acids, the ε differs for each protein. Another disadvantage to this method is the detection limit, which is higher than most other assays including the Bradford and the Lowry assays. Therefore, higher amounts of protein are needed for accurate determination of protein concentration.

Infrared radiation
Infrared (IR) spectroscopy is very useful in determining the concentration and structural properties of many types of proteins including biopharmaceutical proteins. Proteins absorb IR naturally due to characteristic vibrations (stretching and bending) of certain chemical groups along the polypeptide backbone. Measurements of the absorbance of radiation at certain wavelengths can thus be used to quantify the concentration of a protein in the sample.

IR spectroscopy is especially useful for rapid (seconds to microseconds) on-line analysis of protein content. It requires little sample preparation and is nondestructive. Unlike most protein assays, which require protein to be dissolved in a clear solution, IR spectroscopy can be used on gases, solids, and liquids. Its major disadvantages are its high initial cost and the need for extensive calibration.

IR spectroscopy is now a powerful tool for formulation optimization, stability studies and quality control of protein drug products in the pharmaceutical industry. Several types of IR spectroscopy have been developed to increase sensitivity and improve ease of use. These include Fourier-transform infrared (FTIR) spectroscopy and near- infrared (NIR) spectroscopy.

Quantitative Amino Acid Analysis
Quantitative amino acid analysis (AAA) is the primary method for protein quantitation. It is considered the “gold standard” in protein assay because it is not affected by protein composition and does not need a calibration curve with a standard protein.

The analysis of proteins using AAA generally involves the hydrolysis of the protein and subsequent separation of the resultant amino acid mixture using chromatography. To improve detection, amino acids are typically labelled with a fluorescent dye either pre- or post-separation before analysis. AAA is considered a gold standard method because it is reproducible and robust. Commercial reagents and standards used are also readily available.

However, AAA is laborious and expensive and, therefore, not suitable for in-process assays. For this reason, the less accurate secondary colorimetric assays are usually preferred.

Mass spectrometry
Unlike all the other detection methods, which quantitate total proteins in a sample, mass spectrometry can quantitate and identify individual proteins. Thus, mass spectrometry has become the technique of choice for quantifying proteins and peptides in low abundance in many biological studies.

Mass spectrometry is normally used in tandem with liquid chromatography or electrophoresis in the separation of compounds in a sample. In this approach, analytes (e.g. small molecules, metabolites, or peptides) are eluted from a reversed phase column attached to a liquid chromatography instrument and converted to gas phase ions by electrospray ionization. Analyte ions are then fragmented in the mass spectrometer, and fragment and parent masses are used to establish the identity and quantity of the analyte. The detection limit for proteins with the mass spectrometer is on a molar basis. The higher the molecular weight, at the same mass, the higher the detection limit will be for the mass spectrometer.

A defining advantage of mass spectrometry, particularly in biomedical research, is its capability to confidently identify thousands of proteins in complex biological samples without prior specification of the analytes to be measured. However, the cost of the instruments can be prohibitive, and setting up an analysis can be time-consuming. There is also a wide variation in the capabilities of different mass spectrometry tandem methods.

Quantitative Differences Between Assays

CONCLUSION

Protein assays are vital tools in protein profiling in many areas of life science research. As such, the decision on the best assay to use at any given stage of the protein characterization process will have a significant impact further down the product development line. This is of utmost important particularly in biomedicine. Some of the factors to consider in the choice of assay are speed, cost, ease of use, sensitivity, reactivity, and, in the case of drug development, high-throughput capability.

REFERENCES

Rossi, O. et al. (2015). Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA). Molecular Biotechnology 57: 84–93. DOI 10.1007/s12033-014-9804-7

Fabian, H. & Mäntele, W. (2006). Infrared spectroscopy of proteins in Handbook of Vibrational Spectroscopy. John Wiley & Sons, Ltd., New York. DOI: 10.1002/9780470027325.s8201

Noble, J.E. & Bailey, M.J.A. (2009). Quantitation of protein in Methods in Enzymology, Volume 463. Elsevier Inc., New York. DOI: 10.1016/S0076-6879(09)63008-1

Noble, J.E., Knight, A.E., Reason, A.J., Di Matola, A., & Bailey, M.J.A. (2007). A comparison of protein quantitation assays for biopharmaceutical applications. Molecular Biotechnology 37: 99–111. DOI 10.1007/s12033-007-0038-9

Brady, P.N. & Macnaughtan, M.A. (2015). Evaluation of colorimetric assays for analyzing reductively methylated proteins: biases and mechanistic insights. Analytical Biochemistry 491: 43–51. DOI:10.1016/j.ab.2015.08.027

Williams, K.M. et al. (2003). An evaluation of protein assays for quantitative determination of drugs. Journal of Biochemistry and Biophysics Methods 57: 45–55.

Schirle, M., Bantscheff, M., & Kuster, B. (2012). Mass spectrometry-based proteomics in preclinical drug discovery. Chemistry & Biology 19: 72–84. DOI:10.1016/j.chembiol.2012.01.002

Rustandi, R. R., Hamm, M., Lancaster, C., & Loughney, J. W. (2016). Applications of an automated and quantitative CE-based size and charge Western blot for therapeutic proteins and vaccines in Capillary Electrophoresis of Proteins and Peptides, Ch. 16, pp. 197–217. DOI:10.1007/978-1-4939-4014-1_16

Gillette, M.A. & Carr, S.A. (2014). Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nature Methods 10(1): 28–34. DOI:10.1038/nmeth.2309

Zhang, G. et al. (2010). Protein quantitation using mass spectrometry. Methods in Molecular Biology 673: 211–222. DOI:10.1007/978-1-60761-842-3_13.