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Quantification of antibody coupled to magnetic particles by targeted mass spectrometry

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Abstract

Quantifying the amount of antibody on magnetic particles is a fundamental, but often overlooked step in the development of magnetic separation-based immunoaffinity enrichment procedures. In this work, a targeted mass spectrometry (MS)-based method was developed to directly measure the amount of antibody covalently bound to magnetic particles. Isotope-dilution liquid chromatography-tandem MS (ID-LC-MS/MS) has been extensively employed as a gold-standard method for protein quantification. Here, we demonstrate the utility of this methodology for evaluating different antibody coupling processes to magnetic particles of different dimensions. Synthesized magnetic nanoparticles and pre-functionalized microparticles activated with glutaraldehyde or epoxy surface groups were used as solid supports for antibody conjugation. The key steps in this quantitative approach involved an antibody-magnetic particle coupling process, a wash step to remove unreacted antibody, followed by an enzymatic digestion step (in situ with the magnetic particles) to release tryptic antibody peptides. Our results demonstrate that nanoparticles more efficiently bind antibody when compared to microparticles, which was expected due to the larger surface area per unit mass of the nanoparticles compared to the same mass of microparticles. This quantitative method is shown to be capable of accurately and directly measuring antibody bound to magnetic particles and is independent of the conjugation method or type of magnetic particle.

Schematic illustration of the isotope-dilution mass spectrometry-based workflow to directly measure antibody bound to magnetic particles (MP)

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Acknowledgments

We acknowledge the support of the Maryland NanoCenter and its AIMLab. We also thank Dr. Sz-Chian Liou for his assistance with imaging the magnetic particles by TEM. We acknowledge the support of the Professional Research Experience Program (PREP) through the University of Maryland, College Park and the National Institute of Standards and Technology.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20742, USA

    Nicole A. Schneck & Sang Bok Lee

  2. Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899, USA

    Nicole A. Schneck, Karen W. Phinney & Mark S. Lowenthal

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  1. Nicole A. Schneck
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  2. Karen W. Phinney
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  3. Sang Bok Lee
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Correspondence to Sang Bok Lee or Mark S. Lowenthal.

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Certain commercial equipment, instruments, and materials are identified in this paper to specify the experimental procedures and analytical methods adequately. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.

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Schneck, N.A., Phinney, K.W., Lee, S.B. et al. Quantification of antibody coupled to magnetic particles by targeted mass spectrometry. Anal Bioanal Chem 408, 8325–8332 (2016). https://doi.org/10.1007/s00216-016-9948-3

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  • DOI: https://doi.org/10.1007/s00216-016-9948-3

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