Proteins provide structure to cells, catalyse enzymatic reactions, transport cargo, replicate DNA, metabolise nutrients among an array of other functions. Composed of amino acids and generally featuring a three-dimensional structure, proteins form key therapeutic and diagnostic tools as antibodies or peptides. They are intensely studied, designed and manufactured, and can be purified from host cells or tissues and analysed using methods such as Western blotting, X-ray crystallography, a variety of different microscopic techniques, as well as mass spectrometry (MS).
For all these analyses and for manufacturing (in the case of antibodies, for example), ensuring protein purity is key to achieving reproducible results. An impure sample can add significant background noise or produce off-target, unsafe effects when administered in vivo. It is important that proteins are pure and homogeneous, that their concentration is confirmed precisely, and that they are completely in solution, in a natively active state (1).
This article focuses on three main areas important to assessing protein purity that encompass a variety of assays.
Protein purity and integrity
As a first step, purity and integrity must be assessed. This can be done in a simple and fast manner with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), which can also be stained with coomassie blue, a triphenylmethane dye that can help visualise proteins in the electrophoresis gel. Reduction and denaturation with SDS allow protein samples to be assessed for their molecular mass, although sensitivity can be limited.
Other colorimetric dyes such as reverse and silver staining methods can be used to achieve greater sensitivity in visualizing proteins. Fluorescent dyes such as Nile red and CyDyes can be more expensive.
To measure protein concentration, UV-visible (UV-vis) spectrometry can also be used, with a high 260/280 nm absorbance ratio indicating nucleic acid contamination. SDS-PAGE can be followed by mass spectrometry (MS) for more detailed follow-up analysis, although if pre-staining gels with silver stain and then using the glutaraldehyde-free modified method is recommended, as well as avoiding Nile red, which is incompatible with MS.
Capillary electrophoresis can be used instead of SDS-PAGE. This is compatible with MS applications. MS is not only far more sensitive than the other methods mentioned above (at picomole concentrations, it can provide molecular mass measurements for masses up to 500 kDa), it can also detect the presence of post-translational modifications (e.g., phosphorylations, acetylations, ubiquitinations, glycosylations) (1).
Homogeneity, folding and stability
To ensure best results homogeneity of protein samples is important. Here, sample buffer can play a critical role–measures such as pH, salinity, as well as the presence of additives, co-factors or ligands.
To determine homogeneity, dynamic light scattering (DLS) can be used as a simple-to-use technique, detecting uniformity in the size and shape of proteins (monodispersity) as well as high-order assemblies and low amounts aggregates (2). UV-vis can also be used to detect aggregates (with hydrodynamic radio greater than 200 nm) when measuring absorbance over 320 nm. With fluorescent microscopy, excitation at 280 nm followed by measuring fluorescence emission signal at 280 nm and 340 nm provides a ratio (I280/I340) that provides information about the amount of aggregation in the sample.
To best detect oligomerisation, size exclusion chromatography (SEC) can be used to determine the hydrodynamic radius of proteins in a sample. Static light scattering (SLS) by contrast provides molecular mass information and is also sensitive to low levels of aggregates. Ultra-high performance liquid chromatography (UHPLC) can now be used in conjunction with SLS.
SEC should be the last step in protein purification to remove protein aggregates (1). To confirm protein folding, circular dichroism spectroscopy, UV-vis, nuclear magnetic resonance and differential scanning fluorimetry can be used.
In addition, websites that predict protein folding in silico include RF-Fold, SSHMM, THREADER, BLASTLINK, SSEARCH, PSI-BLAST and HMMER (3). Note that for x-ray crystallography, the presence of imidazole, potentially introduced during protein elution, can affect the technique, and should therefore be reduced in concentration, for example via SEC or dialysis (3).
Pure, homogeneous samples should be studied to confirm activity and anticipated function. This can be assessed using functional assays tailored to the specific protein and its range of functions, that assess the percentage of total protein in the sample that is functional.
Total protein concentration can be determined using Bradford, bicinchonic acid (BCA) and Lowry assays, although the composition of control proteins used to draw the standard curve should closely match that of the samples. If the extinction coefficient is known or can be calculated (likely at 280 nm), and the protein in question has a known amount of tryptophans and tyrosines, then UV-vis or Fourier-transformed infrared spectroscopy (FTIR) can also be used for this purpose (1).
Surface plasmon resonance (SPR) can be used to measure the concentration of active, binding proteins, including sandwich SPR, where a protein is first bound to an immobilised tag on a surface, and then the respective ligand is added, and the amount of bound ligand assessed.
Paul Belcher PhD, Product Strategy Manager at Cytiva, shares his expert insights on assessing protein activity: “A ligand binding assay (LBA) is an assay that relies on the binding of ligand molecules to receptors, antibodies or other macromolecules, and are commonly used to confirm protein activity. LBAs include ELISAs, radioligand assays, fluorescence polarization and SPR. Functional assays on the other hand are experiments that are designed to determine the involvement of each protein in a particular cellular pathway or biological process.”
Belcher also says that SPR is extremely useful in not only confirming protein activity via “yes/no binding,” but also in determining the affinity and kinetics of the interaction of interest. “Kinetics allow us to link structure to function and gain a greater understanding of biological mechanisms. When used as a quality control metric, kinetics allows us to see if something has altered in our sample (e.g. change in glycosylation state or structure due to denaturation).”
He says SPR can also be used to determine active concentration–the percentage of the sample that is functionally active (versus total concentration). Typically, in sandwich assays, the molecule/antigen of interest is typically “sandwiched” between two layers, a capture molecule and a detection molecule that is specific to the antigen of interest.
“Since SPR is a label-free method, it doesn’t require a detection molecule (like a fluorescently labelled antibody) – so SPR sandwich assays only need to be employed in specific applications, e.g., epitope binning or to confirm identify/specificity of an interaction.”
SPR being an incredibly useful and ubiquitous technology, Belcher advises that if a smaller research group does not have immediate access to SPR, they could access the technology by collaborating with larger groups or at core analytical service labs within the company or institution.
With such a vast number of techniques and methodologies available to assess protein purity, it is important for scientists to bear in mind what the end application of the protein in question will be, whether in research or for manufacturing purposes. With the end in mind, they can determine what type of analyses and data will be required to confirm purity to the required standard.
- Raynal, B., et al., 2014. Quality assessment and optimization of purified protein samples: why and how? Microbial Cell Factories 13:180.
- Nobbmann, U., et al., 2007. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol Genet Eng Rev, 24:117–128.
- Bhat, E.A., et al., 2018. Key Factors for Successful Protein Purification and Crystallization. Global Journal of Biotechnology and Biomaterial Science.