SISCAPA

Method Description:

SISCAPA, Stable Isotope Standard Capture with Anti-Peptide Antibodies, is a term introduced by Leigh Anderson.1 As shown in the workflow below (Fig. 1), SISCAPA is a hybrid method, combining immunoprecipitation (IP) and stable isotope dilution multiple reaction monitoring (SID-MRM) mass spectrometry to isolate and quantify proteins. Here at the Broad Institute, we’ve adopted and optimized this method to measure biomarker candidates directly from plasma.2

In brief, we immunize rabbits with peptide sequences that are unique for a specific protein and ionize well on a mass spectrometer. After a 77 or 118 day protocol, polysera with a positive titre are antigen purified. Samples are digested into peptides prior to antibody enrichment. assay standardization is accomplished at either the protein or peptide stage by the addition of a stable isotope labeled standard. After IP, the mixture of enriched peptides are injected onto a nano scale reversed phased liquid chromatography column and analyzed on a triple quadrupole mass spectrometer (TQ-MS) using an MRM experiment. Compared to other types of mass spectrometers in the Carr lab, the TQ-MS scans with a 100% duty cycle, which provides the highest level of sensitivity and most precise quantitative measurement. MRM scans can be timed to match the peptide retention time to enable 100’s of peptides to be measured in a single run.

Performance Specifications:

On average, molar amounts of peptide/protein are determined with precision less than 20% across 4 orders of concentration (1ng/ml to 1µg/ml).

Current Focus:

SISCAPA can be multiplexed, i.e. antibodies specific to a number of peptides can be added to the same sample without loss in performance (Fig. 2). Recently data suggests that up to 30 antibodies (or more) can be multiplexed. In order to increase throughput further, strides are being made to demonstrate this method works similarly in plates to process up to 100 samples per week.

Applications:

SISCAPA is primarily used to increase the throughput and detection limits of biomarker candidates as part of the Broad Institute’s Proteomics Platform biomarker verification program. Like other immunoprecipitation techniques, it can also be applied to detect changes in other biological systems (cells, tissues, etc).

References:

1. Anderson, N.L., T.W. Pearson, et al. (2004) "Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)." Proteomics 3(2): 235-44. Abstract

2. Kuhn, E., T. Addona, et al. (2009) "Developing multiplexed assays for troponin I and interleukin-33 in plasma by peptide immunoaffinity enrichment and targeted mass spectrometry." Clin Chem 55:1108-17. Abstract