The SISCAPA Method for Quantitating Proteins and Peptides in Plasma

PPI is exploring the use of a novel method (denoted SISCAPA) for quantitation of peptides in complex digests.   In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence.   Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled).  SISCAPA is thus limited to sequence-defined (predetermined) analytes, but offers the possibility of greatly increased sensitivity (by removing unwanted peptides from the set delivered to the MS).

SISCAPA is described in two initial papers describing 1) the overall method and 2) the techniques for generating and characterizing immobilized Ab supports:

Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). Anderson, N.L.,   Anderson, N.G., Haines, L.R., Hardie, D.B., Olafson. R.W., and Pearson, T.W.   Journal of Proteome Research, in press (2004).

An effective and rapid method for functional characterization of immunoadsorbents using POROS® beads and flow cytometry.   N. Leigh Anderson, N.L., Haines, L.R. and Pearson, T.W. Journal of Proteome Research, in press (2004).

In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin,   a1-antichymotrypsin, interleukin-6 and tumour necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components.   Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS® supports.   Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry.   The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%.   Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays.   The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.

To facilitate the construction, functional characterization, and use of immunoadsorbents, we have developed a flow cytometry method that allows rapid assessment of large numbers of particle-bound antibodies. Protein G derivitized POROS beads were used to bind affinity-purified antibodies specific for synthetic peptides designed from human plasma proteins. The antibodies were covalently coupled to the beads and used to capture and release synthetic peptides that had been labeled at the C-terminus with the fluorochrome Alexa Fluor 488. Antibody coupling and specificity of antigen binding and release were measured by analysis of the POROS affinity beads by flow cytometry. The affinity-capture matrixes were also used through several antigen-binding and release cycles without loss of peptide binding efficiency. The ability to produce and characterize extremely small amounts of POROS affinity matrices will facilitate their use in protein microchemical procedures such as protein chip technology, monoclonal antibody screening and mass spectrometry, applications where analytes are limiting or present in low abundance in complex mixtures.