The SISCAPA Process
The SISCAPA protein quantitation process utilizes trypsin digestion of the sample, followed by a proprietary combination of immuno-enrichment of highly selective anti-peptide antibodies and specific mass spectrometry quantitation.
1. Using a proteolytic enzyme such as trypsin, each target protein is cleaved to yield peptides, many of which have sequences unique to the target (i.e., "proteotypic" peptides). Protein-protein interactions, the source of most immunoassay interferences, are eliminated. The resulting peptides are ideally suited for analysis by MS/MS.
2. A stable isotope labeled version of the signature peptide, easily made by chemical synthesis, is added at known concentration to serve as an internal standard (IS). This standard behaves identically to the signature peptide in affinity or LC fractionation, but is easily distinguished from it in the mass spectrometer.
3. A specific anti-peptide antibody captures the signature peptide and IS, enriching them and depleting other digest peptides, while preserving the signature:IS ratio. Extensive washing depletes matrix peptides, yielding highly purified analytes.
4. Enriched signature and IS peptides are resolved by a short RPLC separation and quantitated in the mass spectrometer by MRM. Given the known concentration of the IS and the measured signature:IS ratio, the signature peptide concentration is calculated.