Perfinity Optimized Columns
Affinity Selection. Each Perfinity Optimized column performs a step of the protein-separation and mass-spec sample-preparation process. Immune complexes are fished out of solution using affinity columns (Perfinity G, Tetravidin, and Monavidin).
The affinity columns reduce the complexity of serum samples down from tens of thousands to three to five components. Proteins are then transferred to a second column that operates in a different mode. Various immunoassays can be performed on subsequent runs by using a different antibody while the system hardware and operation conditions remain the same. The affinity column used depends on the application.
Buffer Exchange. Recent successes in proteomics are based on the fact that proteins are reduced to more easily identifiable peptide fragments by cleavage with proteolytic enzymes, the most popular being trypsin. The catalytic efficiency of trypsin is approximately a million-fold less at pH 2 than it is at pH 8. In a pH 2 mobile phase, proteins are in a solution that is too acidic for trypsin to function. As such, the pH must be adjusted to about 8 before trypsin digestion will occur. After low pH desorption from the affinity column, the Perfinity Buffer Exchange column alters pH to 8 prior to digestion.
Digestion. 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. When more trypsin is used per mass of protein, trypsin begins to autodigest, thereby contaminating the sample and analysis with trypsin fragments.
The Perfinity Trypsin column contains immobilized trypsin. Immobilization prevents autodigestion, making it possible to use a large excess of trypsin. This shortens digestion times from 18–24 hours to 3–5 minutes. As peptides elute from the trypsin column they are concentrated onto the front of the desalting column (Figure 3).
Desalting. In addition to re-concentrating peptides following digestion, the Perfinity Desalting column allows for the removal of salts that negatively affect UV and mass-spec measurements. Samples are directly transferred to a reverse-phase column.
Reverse Phase. A set of high-pressure pumps separates peptides using traditional reverse-phase chromatography. A transfer line can be connected directly to a mass spectrometer.
The Perfinity Workstation automates and integrates affinity selection, buffer exchange, digestion, desalting, and reverse-phase separation steps such that users start with serum and have purified peptides ready for mass-spectral analysis in approximately 10 minutes. Perfinity Optimized columns perform each step of the sample-preparation process while software automates various methods according to application area.