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.