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Hemoglobin depletion from plasma: Considerations for proteomic discovery in Sickle Cell disease and other hemolytic processes

Authors

  • Lisa M. Williams,

    1. Department Pediatrics, Division of Pediatric Hematology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Zongming Fu,

    1. Department Pediatrics, Division of Pediatric Cardiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Pratima Dulloor,

    1. Department Pediatrics, Division of Pediatric Cardiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Timothy Yen,

    1. Department Medicine, Division of Cardiology, Department of Biological Chemistry and the Johns Hopkins Bayview Proteomics Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Emily Barron-Casella,

    1. Department Pediatrics, Division of Pediatric Hematology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • William Savage,

    1. Department Pediatrics, Division of Pediatric Hematology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Jennifer E. Van Eyk,

    1. Department Medicine, Division of Cardiology, Department of Biological Chemistry and the Johns Hopkins Bayview Proteomics Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • James F. Casella,

    1. Department Pediatrics, Division of Pediatric Hematology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Allen Everett

    Corresponding author
    1. Department Pediatrics, Division of Pediatric Cardiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
    • Johns Hopkins University School of Medicine, Division of Pediatric Cardiology, 720 Rutland Ave Ross Bldg, Room 1114, Baltimore, MD 21205, USA Fax: +1-410-955-0897
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Abstract

Purpose: Hemoglobin (Hb) depletion with nickel affinity chromatography has been shown to increase the number of proteins identified in proteomic studies of erythrocytes, but limited data exist on the application of this technique in depletion of Hb from plasma or serum required for clinical biomarker studies. The aim of this study was to explore the potential of using nickel-beads for Hb depletion of plasma.

Experimental design: Nickel–nitrilotriacetic acid (Ni–NTA) affinity chromatography was used to deplete Hb from hemolyzed plasma samples obtained from children with sickle cell disease (SCD, n=7) and normal human plasma (n=4). Ni–NTA-bound proteins were analyzed by one-dimensional GE, followed by in-gel digestion for characterization using an LTQ-Orbitrap hybrid mass spectrometer. In addition, the loss of two non-Hb-related plasma proteins, thrombospondin1 and L-selectin, by Ni–NTA was determined by ELISA (SCD n=6, non-SCD controls n=2).

Results: Ni–NTA resulted in an average 60% decrease in plasma protein concentration, which was not hemolysis dependent. Specifically, Hb (7 peptides) and the top three proteins, α-2-macroglobulin (75 peptides), apolipoprotein B-100 (73 peptides), and albumin (42 peptides) were Ni–NTA bound. In addition, using an ELISA assay two non-Hb-associated plasma proteins thrombospondin1 and L-selectin were decreased by Ni-NTA.

Conclusions and clinical relevance: Hb depletion with Ni–NTA is effective for Hb removal but is not specific. There is a potential for deleterious depletion of potential biomarkers that may limit the applicability of this method. Consideration of alternate methods of Hb depletion for clinical proteomics may be warranted.

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