Key Protein Concentration & Enrichment Techniques

Protein Structure (8).jpg What is protein enrichment and why is there a need for it? Basically, protein enrichment is a technique where proteins of interest in a biological sample are concentrated to make them more suitable for identification and downstream analysis.

In most cases, it is not enough that you have successfully isolated the protein fraction from the solution and the rest of the interfering components. You still need to increase the concentration of your protein of interest and/or reduce sample complexity to make them ideal for further analysis. This is especially important in cases where the total protein content is very low and/or when you are working with minor proteins that have gone through some post-translational modifications.

Protein Enrichments Methods

Several methods are commonly used for protein enrichment. These include centrifugation, precipitation, electrophoretic methods and chromatographic methods.

Centrifugation

Centrifugation is considered the simplest method for protein enrichment and fractionation. It can be used to:

Separate cell substructures in cases where the protein of interest is locally concentrated (e.g. in the membrane, mitochondria or nucleus). By using this method, you can effectively separate the cellular homogenate into its individual components based on molecular weight, size and shape.
Fractionate protein mixtures. Centrifugation can also be used to fractionate protein mixtures based on their coefficient of sedimentation. Basically, the smaller the coefficient value (expressed in Svedberg units), the slower the movement of the molecule in a centrifugal field. Upon centrifugation, the protein particles will separate based on protein density, mass and shape. You can further enhance the efficiency of this step with gradient centrifugation.

Precipitation

Proteins can also be purified and enriched through precipitation. In this method, high amounts of a neutral salt (e.g. ammonium sulphate, sodium chloride) is added to the solution to enhance interactions which then leads to the aggregation and precipitation of proteins.

Generally, ammonium sulphate can precipitate most proteins at saturation (3.9 M at 0^oC or 4.04 M at 20^oC) while preventing bacterial growth and denaturation. The salting-out process can also be manipulated to facilitate selective protein separation by simply varying the salt concentration.

Since antigens have a natural affinity for their specific antibodies, low-abundant protein targets can also be purified and enriched through immunoprecipitation. When using this method, the protein of interest is incubated with the solid-phase beads containing the binding protein. This method is especially useful for purifying and enriching food allergens.

Electrophoretic Methods

Generally, electrophoresis is useful for separating mixtures of proteins based on size, shape, charge and charge-to-mass ratio, and is primarily used as a pre-fractionating technique for one-dimensional separation. Some of the most common electrophoretic pre-fractionation methods include electrokinetic methodologies which rely on isoelectric focusing (IEF) steps. These methodologies allow the proteins to remain in their native conformation so they are ideally used when protein bioactivity must be preserved.

Different instruments have been developed based on the IEF principle. These include the following:

Rotofor - a multicompartmental device that can be used for both initial and final purification steps. The Rotofor allows the fractionation of gram quantities of protein and is capable of enriching low-abundance proteins.
Off-Gel IEF - a novel IEF procedure using a multi-well format that separates proteins based on their pl and allows for the easy recovery of purified proteins directly in solution. Using this methodology, the sample is placed in a liquid chamber positioned on top of an IPG gel which buffers a thin layer of the solution in the liquid chamber. When an electric field is applied perpendicularly to the liquid chamber, the electric field penetrates into the channel and extracts charged proteins into the gel, leaving the globally neutral species in solution.
Octopus - a continuous free-flow IEF device in an upward flowing liquid stream
Gradiflow – a multifunctional electrokinetic membrane apparatus used to purify proteins based on their differences in size, mobility and pl. The separated fractions can be analyzed directly by MS or undergo a subsequent separation step (i.e. SDS-PAGE).

Chromatographic Methods

Chromatography is commonly defined as a physical method of separating individual components (solutes) of a mixture between the stationary phase and the mobile phase. The differences in partition coefficients of the different molecules cause them to separate in the stationary phase.

Liquid chromatography (LC) separates different proteins according to their size, charge, hydrophobicity, or specificity, and is the most commonly used technique in proteome pre-fractionation. It can also be used to remove some interference substances (e.g. salts) which may have been carried through from previous enrichment steps.

Ion-exchange chromatography (IEX), which separates proteins according to their pl, is the most commonly used LC fractionation technique. Anion-exchange chromatography is ideally used in fractionating acidic proteins while cation-exchange chromatography is useful in fractionating basic proteins.

Reverse phase LC (RP-LC) separates proteins based on hydrophobicity. In RP-LC, the mobile phase is slightly more polar than the stationary phase. The hydrophobic molecules in the mobile phase are adsorbed in the stationary phase while the hydrophilic molecules are eluted with increasing concentration of an organic solvent (e.g. acetonitrile). RP-LC is widely used in combination with IEX and MS analysis as an alternative to 2D-PAGE technology.

Affinity chromatography (AC) utilizes highly specific biological interactions to investigate posttranslational modifications (e.g. glycosylation and phosphorylation). Since AC binds high abundance proteins to the column, it can be used to access low concentrated proteins in complex samples.

Immobilized metal affinity chromatography (IMAC), which is based on formation of coordinate bonds between basic groups on protein surface and metal ions, is mainly used for enriching phosphoproteins. Some of the disadvantages of using this technique includes the fact that there is minimal binding to Fe(III) or Ga(III) charged resins at neutral pH, and that using low-pH buffers may trigger protein denaturalization or precipitation in the column.

Size-exclusion chromatography (SEC) separates proteins according to their molecular mass. This technique can be performed under non-denaturing conditions so you can use it in studying protein complexes.