Hemoglobin depletion from plasma: Considerations for proteomic discovery in Sickle Cell disease and other hemolytic processes
Article first published online: 22 NOV 2010
Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
PROTEOMICS - Clinical Applications
Special Issue: Focus on Pathways to Translation of Proteomics into Clinical Practice
Volume 4, Issue 12, pages 926–930, December 2010
How to Cite
Williams, L. M., Fu, Z., Dulloor, P., Yen, T., Barron-Casella, E., Savage, W., Van Eyk, J. E., Casella, J. F. and Everett, A. (2010), Hemoglobin depletion from plasma: Considerations for proteomic discovery in Sickle Cell disease and other hemolytic processes. Prot. Clin. Appl., 4: 926–930. doi: 10.1002/prca.201000054
- Issue published online: 29 NOV 2010
- Article first published online: 22 NOV 2010
- Accepted manuscript online: 5 OCT 2010 09:10AM EST
- Manuscript Accepted: 10 SEP 2010
- Manuscript Revised: 31 AUG 2010
- Manuscript Received: 21 JUN 2010
- National Heart, Lung and Blood Institute (NHLBI). Grant Numbers: U54HL090515, 5R01HL091759
- National Institutes of Health (NIH). Grant Number: U54RR023561
- Hemoglobin depletion;
- Plasma proteomics;
- Sickle Cell disease
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.