Kicking thte Tires
A key quality metric for any automated workflow is the variability of the process. We assessed the performance of the Perfinity Workstation by measuring the variation occurring at each step of the process.
Affinity Capture/Automated Pull-downs.The variability of affinity selection is reduced by enhancing binding kinetics and optimizing recovery (Figure 1).
With the Workstation, affinity selection can be performed in the sample vial such that immune complex formation occurs in solution or in the column (forcing the target through a tunnel of immobilized antibody). The advantage of solution-based incubation is that this system functions in the same way as your immune system, uninhibited by immobilization.
While almost any affinity selector can be used, the use of biotinylated polyclonal antibodies is preferred. Polyclonals typically have higher binding constants, and while prone to cross-reactivity, the loss in selectivity is compensated for by leveraging the resolution of the reverse-phase chromatography performed prior to analysis. What may be the largest advantage of polyclonal antibodies is that they enable you to pull down whole classes of proteins such that all isoforms of a given protein are being captured.
Enrichment. The purification process involves the removal of hundreds of thousands of protein components. Non-specific binding in this type of format is always going to be a concern. However, adsorption is a very weak binding interaction (Kbs of ~104M), while antibodies bind very strongly to antigens (Kbs of ~109M). High degrees of rapid washing enable users to remove proteins that nonspecifically bind (adsorb to) to the antibody.
Recovery. When antibodies bind, it is the result of immunologically tuned interactions that utilize the varying properties of the 20 amino acids (hydrophobicity and charge oriented in an optimized 3-D structure) to generate binding pockets. Recovery is based on the ability to rapidly eliminate the cooperative binding properties of these interactions. Perfinity Optimized Buffers account for the wide variety of interacts that influence antibody binding, allowing for high levels of recovery and reproducibility.
Automated Trypsin Digestion. A dizzying number of interactions affect traditional solution based trypsin digestion. Many of the problems and limitations of solution-based digestion can be eliminated by rapidly pushing the reaction to completion.
Trypsin digestion is most widely achieved by incubating the protein mixture with a 50:1 mass ratio of protein:trypsin for a 24-hour period (Figure 2). When more trypsin is used per mass of protein, trypsin begins to autodigest contaminating the sample fragments and reducing enzyme activity.
The Perfinity Trypsin Column contains immobilized trypsin. Immobilization prevents autodigestion making it is possible to use a large excess of trypsin enabling rapid, complete digestions. Hydrophobic domains buried in the structure of the whole protein are exposed during digestion resulting in peptides of low solubility. Recovering these peptides is optimized to enhance solubility without impacting reverse-phase retention. Early on in the development of our system, carryover greatly affected the reproducibility of our results, but further optimization of the Perfinity Digest Buffer eliminated this problem.
Buffer exchange and desalting. The immunoaffinity LC/MS/MS process also includes a buffer-exchange step where eluent buffer is exchanged for digest buffer. Lyophilization, dialysis, and spin column techniques are all prone to sample loss due to the surface interactions and the open nature of these systems. Also, when proteins are eluted from affinity-based capture columns, they are denatured exposing hydrophobic domains in a similar manner as described previously.
Many research groups have struggled with recovering denatured proteins that have had the opportunity to interact with surfaces. Keeping this in mind, Perfinity created a flow-through column containing a hydrophilic skin that repels proteins, and the Perfinity Digest Buffer was optimized to reduce the impact of electrostatic forces between charged amino acids ensuring quantitative transfer from between steps.
The final step prior to LC/MS/MS analysis is the desalting of dilute peptides. Given the dilute nature of the peptides being eluted from the enzyme reactor it is important that the column material be hydrophobic enough to retain diluted peptides without being overly hydrophobic resulting in retention that would exceed that of the analytical column and produce band spreading. These considerations came together resulting in a system with low variability (CVs below 10% for a five-step process.
Protein analyses involve high levels of analyte and sample complexity. Quality results are dependent on an understanding of binding kinetics, recovery, solubility, activity, retention, and a variety of other factors. Key to the value of the Perfinity Workstation format is accounting of these factors through an integration of columns, buffers, and software enabling users to shift their focus from workflows to answers.