The patented "Stable Isotope Standards and Capture by Anti-Peptide Antibodies" (SISCAPA) workflow consists of a series of simple liquid addition steps followed by magnetic bead extraction, and is ideally suited for implementation on a laboratory robot operating in a 96-well plate format. The generic workflow can be used for a range of protein analytes in single or multiplex assays, and on a variety of sample types.
1. A stable isotope labeled version of the selected signature peptide, made by chemical synthesis, is added at known concentration to serve as an internal standard (SIS). This standard behaves identically to the signature peptide in affinity or LC separations, but is easily distinguished from it in the mass spectrometer.
2. Using a proteolytic enzyme such as trypsin, sample proteins including the target protein are cleaved to yield peptides, including the signature peptide. Protein-protein interactions, the source of most immunoassay interferences, are eliminated.
3. An anti-peptide antibody specific for the selected peptide sequence captures the signature peptide and IS, enriching them away from other digest peptides, while preserving the signature:IS ratio. Extensive washing depletes matrix peptides, yielding highly purified peptide analytes.
4. Enriched signature and IS peptides are resolved by a short LC separation (if necessary) and quantitated in the mass spectrometer, typically by MRM. Given the known concentration of the IS and the measured signature:IS ratio, the signature peptide concentration is calculated.
- a capture reagent that enriches the specific proteotypic peptide to be measured (a monoclonal antibody)
- a labeled version of the peptide that serves as an internal standard for MS quantitation
Everything else (workflow and instrumentation) can be constant for all assays, allowing true mix-and-match multiplex panels.
Automated SISCAPA protocols, available from SAT, have been developed for Beckman Biomek NXP and Agilent Bravo liquid handling robots. These protocols make use of an ‘addition only’ tryptic digestion protocol that eliminates the need for sample cleanup (e.g., SPE, filtration, centrifugation), while improving reproducibility, scalability and running cost. In most cases these protocols process one or two 96 well plates of samples in ~4 hr, generating a plate of enriched target peptides ready for delivery to the mass spectrometer.
This step-by-step instructional video describes how the SISCAPA assay protocol has been implemented on a highly automated robotic platform (Agilent's Bravo in this case) for high precision and high throughput with minimal user intervention.
A ‘pool and split’ experiment was performed to separately measure the imprecision attributable to each stage of the workflow. Thirty-six replicate samples (equal aliquots of a single pooled human plasma sample) were digested with trypsin in three rows of a 96-well microtiter plate divided into three sets of 12 samples (N = 36). Samples in the first set were pooled at the end of the SISCAPA peptide enrichment process (immediately before MS analysis), mixed and redistributed into 12 equal aliquots. These samples exhibit the imprecision due to the MS analysis alone. Samples in the second set were pooled after the digestion step (just before addition of heavy peptides and the SISCAPA peptide enrichment process), mixed and redistributed into 12 equal aliquots. These samples exhibit the imprecision of SISCAPA enrichment + MS analysis. Samples in the third set were not altered in any way during the process and exhibit the imprecision of the entire workflow (i.e., Digestion + SISCAPA peptide enrichment + MS analysis). The entire ‘pool and split’ experiment was performed on three separate days.
Using an automated liquid handling system, tryptic digestion and SISCAPA enrichment steps do not add appreciably to overall % CV, representing <3% for the three peptides examined here.
CV's of Peak Area Ratios