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Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid

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Abstract

Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 μL) due to low yields stemming from losses caused by nonspecific binding to the column matrix and concentration of large eluent volumes. Additionally, the cost of the depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 μL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ∼6-μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-μL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.

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Acknowledgments

This project was supported by grants from the National Center for Research Resources (5 P41 RR018522-10) and the National Institute of General Medical Sciences (8 P41 GM103493-10) from the National Institutes of Health as well as the Department of Energy Office of Biological and Environmental Research Genome Sciences Program under the Pan-omics project. Work was performed in the Environmental Molecular Science Laboratory, a U.S. Department of Energy (DOE) national scientific user facility at Pacific Northwest National Laboratory (PNNL) in Richland, WA. Battelle operates PNNL for the DOE under contract DE-AC05-76RLO01830. We appreciate the favor from Jonas Bergquist at Uppsala University (Department of Chemistry-Biomedical Center, Analytical Chemistry and SciLife Lab, Uppsala, Sweden) for the donation of CSF sample.

Conflict of interest

The authors declare no conflicts of interest.

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Authors and Affiliations

  1. Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99352, USA

    Seok-Won Hyung, Paul D. Piehowski, Ronald J. Moore, Daniel J. Orton, Athena A. Schepmoes, Therese R. Clauss, Rosalie K. Chu, Thomas L. Fillmore, Heather Brewer, Tao Liu, Rui Zhao & Richard D. Smith

  2. Korea Research Institute of Standards and Science, 267 Gajeong-Ro, Yuseong-Gu, Daejeon, 305-340, Republic of Korea

    Seok-Won Hyung

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  1. Seok-Won Hyung
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  2. Paul D. Piehowski
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Correspondence to Seok-Won Hyung or Richard D. Smith.

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Hyung, SW., Piehowski, P.D., Moore, R.J. et al. Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid. Anal Bioanal Chem 406, 7117–7125 (2014). https://doi.org/10.1007/s00216-014-8058-3

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  • DOI: https://doi.org/10.1007/s00216-014-8058-3

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