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

• DOI: 10.1074/mcp.m800446-mcp200
• Publication Date: 2009-02-07T04:22:05Z
• Authors: N. Leigh Anderson; Angela Jackson; Derek Smith; Darryl Hardie; Christoph Borchers; Terry W. Pearson
• Keywords: Bead; Magnetic bead

A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based of individual tryptic peptides from a digest whole human plasma was developed using simplified magnetic bead protocol novel rotary trap device. Following off-line equilibrium binding antibodies subsequent the on beads, permitted washing beads elution bound inside 150-microm-inner diameter capillary that forms part nanoflow LC-MS/MS system. The sweeps against direction liquid flow continuous succession moving high field-gradient regions while mixing with flowing liquid. This approach prevents loss low abundance captured allows automated processing series reactions. Selected alpha(1)-antichymotrypsin lipopolysaccharide-binding protein were enriched relative to serum albumin peptide 1,800 18,000-fold, respectively, as measured multiple reaction monitoring. large majority are nonspecifically in reactions shown bind components other than antibody (e.g. beads), suggesting substantial improvement enrichment could be achieved development improved inert surfaces.