Poor quality protein preparations can dramatically impact downstream analyses, confounding results and wasting significant time and money.
For these reasons, it is vital to assess protein quality prior to performing in-depth protein characterization and functional studies.
Before we discuss the implications of poor protein quality, it is first important to understand what factors to consider when assessing protein quality.
What does protein quality mean?
Often a rapid check of concentration by UV is considered a quality assessment, but this is only one piece of the puzzle.
Measuring protein size not only allows confirmation that the correct protein has been selected or generated, it also provides information about protein conformation. For example, it is possible to identify size changes caused by misfolding, oligomerization or aggregation (Figure 1). As we will see later, such conformational changes can have a major impact on protein function.
Protein size can also be used to determine whether the protein has been expressed in a full length or truncated form. Insights can also be gained into the presence of any post-translational modifications.
Ensuring protein purity is essential when conducting downstream analyses. Contaminants such as unwanted proteins or nucleic acids can have a detrimental impact on kinetic or functional analyses, confounding experimental interpretation.
Most protein purification methods are based on chromatography, where differences in the properties of proteins and other substances in the sample are used to separate them, for example, charge, size, hydrophobicity, etc. Affinity chromatography (AC) is commonly the first purification step, where the target protein is tagged with a specific ligand that can be easily extracted from the sample. However, depending on the nature of the sample, other purification methods may also be required.
Accurate protein quantification is fundamental to achieving comparable and reproducible results, which, in turn, is essential to the correct interpretation of analytical data. Even small differences in protein concentration can have a large impact on downstream kinetic analysis and functional studies.
The correct folding and function of proteins can be strongly influenced by a wide range of factors, including temperature, pH, salt concentration, preservatives and cosolutes. Identifying the influence of different buffers on protein stability can be achieved though measuring characteristics such as protein size and concentration.
Again, these are important factors to consider prior to performing in-depth and often expensive protein characterization studies. Read more about the impact of changing buffer and temperature conditions on interleukin-2 aggregation here.
How does poor quality protein impact research?
The use of poor-quality protein can have a profound impact on the outcome of downstream analyses, resulting in substantial time and cost implications. Here we see the 3 key ways that poor protein quality impacts research, underlining the requirement for routine protein quality assessment prior to further analyses.
Impaired protein function
In a research context, poor protein quality can make data interpretation extremely challenging and reduce the validity of results. As discussed above, different protein concentrations, protein folding and purity can all significantly influence protein function and it is vital to measure and control all of these potential experimental variants in order to obtain reproducible, valid results. It is important that the quality of protein samples obtained from both in-house preparations and purchased from external suppliers are routinely assessed prior to experimental usage to ensure lot-to-lot consistency and reproducible results.
In biotech and biopharma, assessment of protein quality is essential to batch reproducibility. For example, insulin is produced as a hexamer in the body; however, it is the much smaller, less stable monomer form that is biologically active. Amyloid aggregates of insulin can cause severe problems for insulin therapy patients as amyloid fibrils build up at injection sites, which can impair insulin absorption. Studies have estimated the cost of excess insulin required by patients with amyloid is approximately $3k per patient. As such, it is highly important to understand and measure protein isomerization and aggregation.
Time to result
The requirement to repeat various downstream protein analysis techniques after initial use of low-quality protein can significantly affect time to result. This is especially evident when using techniques such as Analytical Ultracentrifugation (AUC), which can take up to 24 hours per sample.
The impact on time to result is further compounded when external or shared facilities are required, as is common for cryogenic electron microscopy (cryo-EM), mass spectrometry and antibody generation.
It is clear that performing downstream analysis and functional studies on poor quality proteins will lead to increased costs due to the subsequent requirement to repeat experiments. This may amount to hundreds or even thousands of dollars per sample, depending on the analysis technique or assay utilised. For example, 3D structural analysis of proteins and protein complexes using a cryo-EM service provider typically costs upwards of $2,000 per sample.
Make quality a priority
As we have seen, routine characterization of protein quality prior to more extensive characterisation or use in functional assays is critical. A number of techniques are available for cost-effective analysis of protein size and concentration, each of which differ in their respective strengths and limitations (follow the links for a more detailed overview of these techniques).
The Fluidity One from Fluidic Analytics provides rapid, cost-effective and accurate analysis of protein quality. The platform utilizes microfluidic diffusional sizing (MDS) to precisely measure, protein size, concentration and stability. Samples are analysed in solution under near-native conditions to ensure correct protein conformation. Results are obtained within 8 minutes using just 50 ng of sample, allowing rapid characterisation of precious samples. Find out more about the advantages of Fluidity One here.
- Raynal, B. et al (2014). Quality assessment and optimization of purified protein samples: why and how? Microb Cell Fact. 13: 180.
- Nilsson, M.R. (2016) Insulin amyloid at injection sites of patients with diabetes. Amyloid. 23(3)