Written by Yuning Wang, PhD

What is protein purity?

When scientists refer to protein purity, they are referring to a sample’s properties and homogeneity. Ideally, if a sample contains protein B, the sample should only contain protein B, rather than protein B and x, y, z proteins and non-protein molecules. Furthermore, the homogeneity of a sample refers to the uniformity of that same protein, so additional chains, aggregates, misfolded proteins would not be representative of a pure and homogenous sample. All of these can severely impact the quality, safety, and function of a protein sample.

What Impact Does Poor Protein Purity Have?

Protein purity is critical to the quality of an experiment. In the lab, proteins are typically expressed and purified from expression systems like E. coli. Undesired artifacts (e.g., nucleic acids, carbohydrates, lipids, and non-target – exogenous and endogenous – proteins) can be introduced during the purification process. Such contaminants can lead to poor experimental quality, inaccuracy, and low precision.

Aside from the commonly known artifacts, target proteins can misfold, aggregate, or degrade. In extreme cases, protein aggregation results in the target proteins precipitating out of solution, an often irreversible process. Furthemore, aggregation and degradation can impact optimal protein performance such as poorly binding to other proteins they would naturally interact with. The latter can deeply affect reproducibility.

As such, taking the time to control the factors that influence protein purity is of vital importance. Some conditions such as pH, salt concentration, buffer, storage temperature, and protein concentration can be controlled relatively easily. However, protein quality can alter over time, highlighting the importance of routine testing and monitoring to save on cost, resources, and time.

How to Check Protein Purity

When checking for protein purity, it is important to measure concentration, before assessing other properties such as size, and charge. High concentrations of impure protein fractions can impact protein order, causing them to aggregate or misfold, and even form artifact complexes with other protein impurities. Once the concentration has been measured, and appropriate aliquots have been taken, protein samples can then be analyzed based on size and charge. The latter are critical to differentiating between different proteins, and effectively separating them.

Key Techniques Used to Check Protein Purity

Spectrophotometry and Colorimetric Assays

Measuring the concentration of a protein sample is important because concentration can impact protein aggregation. Ultraviolet-visible (UV-Vis) spectrophotometry – more commonly known as the use of a Nanodrop-like instrument, and colorimetric assays such as Bradford, and BCA assays are the most widely-used methods to measure protein concentration. 

In addition, UV-Vis can also be used for the detection of non-protein contaminants (e.g., nucleic acids), and the presence of large particles (e.g., large protein aggregates) by monitoring the absorbance signals at particular wavelengths1.

Electrophoresis

Electrophoresis is a technique that separates proteins based on their charge and size using electrical input applied to a gel in solution. This can be useful in separating the target protein from non-target proteins which theoretically should bear different physicochemical properties. It is important to have a general idea of the concentration of protein prior to performing electrophoresis as heavily concentrated proteins may precipitate out of solution, aggregates, and/or generally have a difficult time separating in gel. There are many types of electrophoresis techniques, perhaps the most common being polyacrylamide gel electrophoresis.

Dynamic Light Scattering

Dynamic Light Scattering (DLS) is a powerful tool to study diffusion behaviour of proteins based on their size and shape. DLS is often used to probe homogeneity of purified proteins, especially the presence of higher order structures, complexes, and aggregates1. With DSL, the size distribution profile of a protein sample can be determined.

Chromatography

There are many types of chromatography that can be used to separate proteins based on charge, size, and/or their ability to bind a specific protein. Liquid chromatography which includes analytical size exclusion chromatography (SEC) is a routinely used separation technique. SEC, as an example, can be used to detect protein oligomerization and separate different species by their size. 

Aggregates and different molecular contaminants present in the protein sample can also be readily separated and quantified with SEC. The most commonly used type of liquid chromatography is High Performance Liquid Chromatography, commonly known by its acronym HPLC. Finally, affinity chromatography can be particularly useful to purify immunoglobulins prior to checking their purity by HPLC, or the aforementioned methods.

Mass Spectrometry

Mass spectrometry is an extremely powerful analytical method that can be used to identify proteins based on mass and charge. Protein impurities may be easily detectable with the mass spec depending on the instrument’s resolution, the abundance of the impurity, and the acquisition method of the instrument (e.g., data-dependent acquisition vs data-independent acquisition).

Handling Sample Protein Purity at Rapid Novor

When it comes to de novo protein sequencing, Rapid Novor easily manages samples with bovine serum albumin, and other impurities that previously challenged mass spec experiments. However, it is always optimal for a sample to be pure. Below we list some resources about how to get protein samples ready for de novo protein sequencing:

 If you would like to find out more about how to check protein purity or are unsure about the purity of your sample but would still hope to sequence it, please reach out to one of our scientists to discuss sample requirements.

References

  1. 1. Raynal, B., Lenormand, P., Baron, B., Hoos, S. & England, P. Quality assessment and optimization of purified protein samples: why and how? Microbial Cell Factories 13, 180 (2014).

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