Chromatography comprises a group of molecular separation techniques commonly used in proteomics to isolate proteins of interest from heterogeneous samples. Despite the fact that these methods are purification strategies in their own right, it would be ill advised to introduce crude sample to your chromatography setup from the outset. The vast majority of samples encountered require some form of processing before they can be placed on the analytical instrument. For example, any starting material bearing high ionic strength, in addition to electrolyte-rich isolation and extraction buffers, may interfere with ion exchange (IEX) chromatography, resulting in low protein retention. Similarly, any particulates remaining in poorly prepped samples may cause clogging of the column, potentially invalidating your run and wasting precious sample in the process. Such constituents need to be removed before samples even get near a chromatography column, or else you could end up with undesirable or irreproducible results.
There are also several other aspects of sample preparation you should consider. Here we briefly discuss the steps you can take to improve the quality of your chromatography sample prior to performing your column separation, to ensure optimal isolation of your target protein.
Preparation is key
It sounds obvious, but a good starting strategy is to collate as much information as possible on your sample material and specifically the protein of interest. Being clear on the material’s features and properties will enable you to prepare the highest-quality sample while retaining as much of your target protein as possible by keeping the number of preparation techniques required to a minimum. Properties such as the molecular weight, relative abundance and hydrophobicity of your target protein will help you determine which preparation strategies are essential to your workflow. Online bioinformatics databases, such as uniprot.org, are extremely useful in this regard.
Clear things up
A large component of your crude cell or tissue lysate will consist of debris from insoluble biological material and particulates. Centrifugation at 45, 000 rpm for 30 minutes with a 45 Ti rotor or equivalent (in a chilled centrifuge) will isolate the bulk of this. Following this step, your sample will require clarifying further—even the smallest of particulates can clog pores in any chromatography medium; they can also affect hardware such as HPLC flow lines and rotary injection valves. To avoid these pitfalls, pass your sample through a 0.45- or 0.2-µm filter to remove fine particulates. If your sample clogs up in even 0.45-µm filters, you can opt for a 0.8-µm gauge filter instead; you can always switch back to a finer filter after you have refined your sample using the larger pore size. Pressurized or vacuum filtration systems can also be an asset here.
Further regarding your choice of filter product, opt for those with low protein-binding membranes such as those made from polyvinylidene fluoride (PVDF) or polyethersulfone (PES). You want to avoid using membranes with high protein affinities, such as cellulose- or nylon-based membranes, as they will reduce total protein yield significantly and thus the amount of your target protein. An additional tip to consider: If your sample is too viscous, it could be because of contaminating DNA. Add DNase to your sample before filtration to reduce viscosity caused by intact DNA strands, and you should get a smoother flow through the column
Deplete abundant protein species
Fluid biological samples such as blood or serum can have a complex composition. This is compounded by their high ‘dynamic range’: a steep ratio between the most and least abundant protein species present. The dynamic range of fluid biological samples can be somewhere in the range of 10 and 12 orders of magnitude [1]. Take human plasma as an example: Just 22 proteins make up 99% of its bulk mass of total protein. Several hundred thousands of others make up the remaining 1% [2]. If you begin your search for a rare biomarker in this mixture, you may be setting yourself up to fail—a lot like trying to go stargazing in broad daylight. The presence of high-abundance proteins suppresses the signals of those low-abundance ones, and current analytical methods struggle with such contrast; cue sample depletion.
There are several methods for sample depletion available to researchers, ranging from those that remove one or two of the most abundant constituents to those that are a little more complex and extract whole swaths of proteins. One of the most common strategies in the former category is to selectively remove high-abundance proteins such as albumin and immunoglobulin. Commercial kits are available from several tool providers.
Another depletion strategy mixes solid phases with different protein specificities; this mixture of immunosorbent media targets several high- to medium-abundance proteins at once. As there is a set ratio of each type of immunosorbent present, a representative amount of each targeted protein is left in the sample—high-abundance proteins are depleted, and low-abundance targets become enriched. This strategy is also known as protein enrichment, and various kits and reagents are available from a variety of tool providers to assist researchers in streamlining the process.
Clean up your sample
Certain compounds may interfere with your chosen chromatography resin. For instance, contaminating salts interfere with the binding capabilities of IEX chromatography resins, and detergents and lipids can hinder effective separation using hydrophobic interaction chromatography columns. In these situations, adding a sample clean-up step will improve the quality of your downstream results. This step can be as simple as performing dialysis in a lower salinity buffer, but this approach can take several days and buffer changes to complete.
The removal of contaminants may be achieved in a single step via size-exclusion gel filtration, in which proteins are eluted before salts and other small contaminants because they cannot access the interior of the gel matrix. This technique is also known as buffer exchange, and there are several commercially available kits for researchers to use. They provide the advantage of sample elution into a buffer that is appropriate for the next steps in your chromatography workflow.
Concentration of your sample
Although sample-preparation procedures vary, depending on the chromatography technique being utilized, most chromatography experiments require only a small volume of sample; therefore, the samples must have a high protein concentration. Sample concentration is an effective way of loading more protein per analysis and assists with protein-resin binding. Plus, controlling protein concentration will not only ensure the success of individual experiments, it will also help you with reproducibility, if that is your goal (enabling you to load the same concentration of protein per experiment).
If you’re using a method of buffer exchange for contaminant removal, you can elute into a small volume and adjust this accordingly to obtain the concentration required by following a protein assay.
However, if you are handling large volumes, you can use spin columns to concentrate your sample. Keep in mind that membrane pore sizes vary, so carefully select the appropriate spin concentrator for your protein sample and also choose a low protein-binding membrane such as PES, so you don’t lose precious material during the process.
Getting a good start
Chromatography techniques are constantly evolving to meet the demands of the fast-paced proteomics field, yet many of its fundamental challenges can be addressed by proper sample preparation. Though the sample-preparation stage is as crucial as any other step in the chromatography workflow, it doesn’t always receive the care and attention it should. If you have been neglecting this area or are simply looking for a place to start, these hints and tips will set your sample-preparation workflow on the right track.
References
[1] Pieper, R, et al., "The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins," Proteomics, 3(7):1345-1364, 2003. [PMID: 1287236]
[2] Borg, J, et al., "Spectral counting assessment of protein dynamic range in cerebrospinal fluid following depletion with plasma-designed immunoaffinity columns," Clin Proteomics, 8(1):6, 2011. [PMID: 21906361]
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