SISCAPA peptide enrichment on magnetic beads using an in-line bead trap device

Abstract

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

Fig. 1.

Prototype bead trap device. A, side view; B, schematic diagram; C, top view. N, north; S, south.

Fig. 2.

LC-MS/MS plumbing diagram showing integration of bead trap into the fluidic system used. N, north; S, south; IPA, isopropyl alcohol.

Fig. 3.

Elution of fluorescein-labeled peptide from magnetic beads during washing and elution in bead trap.

Fig. 4.

Enrichment of medium abundance AAC-1 peptide. Traces of MRM signals during LC-MS/MS analysis of unfractionated plasma digest (A) and peptides eluted in bead trap from beads carrying anti-AAC-1 antibody (B) are shown. Peak 5 is a major albumin peptide (HSA-1), and peak 3 is AAC-1 peptide; others are identified in Table I. cps, counts/s; XIC, extracted ion chromatogram.

Fig. 5.

Enrichment of lower abundance LBP-1b peptide. A, unfractionated digest using a more comprehensive MRM set; B, isolated LBP-1b MRM trace from A; C, peptides eluted in bead trap from beads carrying anti-LBP-1b antibody; D, peptides eluted off line from beads carrying anti-LBP-1b antibody. Peptide peaks are identified in Table I. cps, counts/s; XIC, extracted ion chromatogram.

Fig. 6.

Bead trap elution of a SISCAPA capture reaction (A) and a mock capture omitting antibody (B). cps, counts/s; XIC, extracted ion chromatogram.