The world rejoiced at the completion of the Human Genome Project. Researchers are now faced with the challenges of the proteome, which is vastly more complex and data rich. The diagnostics industry is expanding upon traditional immunological assays, utilizing tests that discriminate isoforms and splice variants, while at the same time identifying post-translational modifications.
In the pharmaceutical sciences, the industry-wide shift from small molecule therapies to biopharmaceuticals has resulted in drug therapies that are much more difficult to isolate with intricacies that are exceedingly difficult to detect. Mass spectrometers have the capability to resolve these complexities but only when preceded by separation processes that prepare proteins and peptides for analysis. A problem with current fractionation approaches is that they are complex, multifaceted, and often yield irreproducible results.
Perfinity’s new Perfinity Workstation exploits novel separation technology to automate sample preparation. The Perfinity Workstation enables users to start with serum and have peptides ready for LC/MS analysis in as little as 10 minutes. This system dovetails nicely into most existing LC/MS workflows.
The Perfinity Workstation consists of five columns. Each column performs a step of the mass-spec sample-preparation process—affinity selection, buffer exchange, digestion, desalting, and reverse-phase separation. Automated integration of these steps removes much of the equipment and labor associated with mass-spectral analyses of proteins (Figure 1).
Standard immunoassays have been the dominant method of performing routine serum protein analyses for the last 60 years for good reason. They are a conceptually simple method capable of measuring antigens down to the pg/mL level.
The Perfinity approach to separations is an immunoassay technique that replaces the second antibody of a sandwich pair with a chromatography column. The complete system includes trypsin digestion followed by chromatography and mass spectral analysis. There are significant benefits to this approach.
Differences among protein isoforms often occur in small hidden regions of the total structure. Immunological contact areas, or epitopes, are small, making it difficult to interrogate these domains with antibodies. Moreover, antibodies do not cleanly discriminate between single amino acid variants.
Chromatographic retention is generally determined by a relatively large portion of the surface of a protein or peptide. Variants that differ by a single amino acid can be resolved. However, serum cannot be analyzed directly. Proteins must be extracted or samples fractionated prior to analysis.
With immunochromatographic analyses, the structural selectivity of antibodies can be used to bind and purify antigens from biological extracts, then chromatography can be used to resolve proteins that differ by only small changes in structure. In a given run, hundreds of peaks can be resolved, which enables high degrees of multiplexing (Figure 2).
Researchers have found ways to leverage combinations of these features, using multiple apparatuses and transfer pipettes. Buffer exchanges and a solution-based digestion of proteins into more easily identifiable peptide fragments are performed off-line.
The Perfinity Workstation combines the selectivity of antibodies with the resolving power of chromatography. Coupling of affinity columns that select proteins of interest to immobilized trypsin generates requisite peptides, making it easy to distinguish between single amino acid variants via mass-spectral analysis. Buffer exchange and desalting are performed on-line. The reduction in sample-processing time enables users to screen a variety of conditions rapidly.
A major advantage of Perfinity’s sample-preparation approach, resulting from the work of Perfinity founder Fred Regnier, is that unlike other technologies users are not required to immobilize antibodies. Instead, antibodies are added to the sample and allowed to function as they would as part of the immune system—antibody-antigen complexes are formed in solution. This results in enhanced kinetics. Removal of the immobilization process makes for one less possible source of error.
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.
Kevin Meyer ([email protected]) and Nick Herold are research scientists at Perfinity Biosciences.