Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

The advanced immuno-MS work flow for serum biomarker quantification using the antibody-immobilizing magnetic beads (#877)

Yoshio Takahata 1 , Misato Hara 2 , Kouhei Nishino 1 , Naohiro Hanyu 2 , Takao Kawakami 1
  1. Medical ProteoScope Co., Yokohama, Japan
  2. Tamagawa Seiki Co., Iida, Japan

Immuno-MS, mass spectrometric peptide measurement combined with immunoaffinity purification of protein, is a powerful method for quantitative analysis of low abundant proteins in biological specimens. In the procedures it is crucial to collect specifically and efficiently the target protein antigens from the antigen-antibody complex generated on the surface of the nano-scaled carrier beads. The optimized conditions of the immunoaffinity purification include elution of the target protein with an aqueous organic solvent.
As an experimental model, we used serum alpha-fetoprotein (AFP), one of the hepatocellular carcinoma biomarkers, and its specific antibody immobilized covalently on the magnetic beads. The human serum was spiked with known amounts of the standard AFP. The antibody-coated beads were incubated with the spiked serum for antigen-antibody reaction. AFP was then eluted from the antibody-coated magnetic beads with 0.1% TFA aqueous solution containing organic solvents. The eluted protein was hydrolyzed with a trypsin/LysC mixture. Stable isotope-labeled standard peptides were added to the hydrolysate to quantitate the eluted AFP by LC-MS/MS.
Various organic solvents in the elution solution facilitated AFP recovery, which was maximized with an aqueous solution containing 50% acetonitrile and 0.1% TFA. Moreover, using the optimized workflow, quantitative analyses showed a correlation between the amounts of AFP spiked into serum (0–100 ng/ml) and the corrected ion intensities of the tryptic peptides from AFP (R2 > 0.99). The concentration of the endogenous AFP was calculated as 2.3 ± 0.6 ng/ml from the standard curve regression equation. This result is consistent with previous reports for AFP concentration in healthy human sera (< 10 ng/ml). The present immuno-MS workflow is easily applicable to detection and quantitation of the other low abundant biofluid biomarkers.