SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

Abstract

A SISCAPA (stable isotope standards and capture by antipeptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. MS is the method of choice for identification of peptides in digests of biological samples based on the power of MS to detect the chemically well defined masses of both peptides and their fragments produced by processes such as CID. This high level of structural specificity is also critical in improving peptide (and protein) quantitation because it overcomes the well known problems inherent in classical immunoassays related to limited antibody specificity, dynamic range, and multiplexability. In principle, a quantitative peptide assay using MRM 1 detection in a triple quadrupole mass spectrometer should have nearly absolute structural specificity, a dynamic range of ϳ1eϩ4, and the ability to multiplex measurements of hundreds of peptides per sample (1). These properties suggest that MS-based methods could ultimately replace classical immunoassay technologies in many research and clinical applications.An important limitation of present peptide MRM measurements is sensitivity. The most sensitive widely used quantitative MS platforms use nanoflow chromatography and ESI to deliver trace amounts of peptides to the mass spectrometer. However, these processes are limited in the total amount of peptide that can be applied while retaining maximum sensitivity (typically limited to ϳ1 g of total peptide sample, i.e. the product obtained from digesting ϳ14 nl of plasma). The lower cutoff for detecting proteins in a digest of unfractionated plasma by this approach appears to be in the neighborhood of 1-20 g/ml plasma concentration, which would restrict analysis to the top 100 or so proteins in plasma (1).The sensitivity of MS assays can be substantially increased by fractionating the sample at the level of intact proteins, the tryptic peptides derived from them, or both. For example, immunodepletion of the six most abundant plasma proteins, removes ϳ85% of the protein mass (2) and results in an increase of ϳ7-fold in the signal-to-noise of MRM measurements of peptides from the remaining proteins after digestion (1). Similarly chromatographic fractionation by strong cation exchange provides another major improvement in sensitivity (3). However, increased sample fractionation brings with it the disadvantages of increased cost and time, the risk of losing specific components, and the continued requirement for very high resolution (lengthy, low throughput) reversed phase nanoflow chromatography en route to the ESI source.An alternative fractionation approach, used in the SISCAPA method, enriches specific target peptides through capture by anti-peptide antibodies, thus circumventing these disadvantages for preselected targets (4). In its initial implementation, SISCAPA used very small (ϳ10-nl) columns of POROS chro-

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