title: Hemoglobin depletion from plasma: considerations for proteomic discovery in sickle cell disease and other hemolytic processes.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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, -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
  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.
authors.affiliation: Department Pediatrics, Division of Pediatric Hematology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Williams
authors.forename: Zongming
authors.initials: Z
authors.lastname: Fu
authors.forename: Pratima
authors.initials: P
authors.lastname: Dulloor
authors.forename: Timothy
authors.initials: T
authors.lastname: Yen
authors.forename: Emily
authors.initials: E
authors.lastname: Barron-Casella
authors.forename: William
authors.initials: W
authors.lastname: Savage
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
authors.forename: James F
authors.initials: JF
authors.lastname: Casella
authors.forename: Allen
authors.initials: A
authors.lastname: Everett
mesh_terms: D000293:Adolescent; D000755:Anemia, Sickle Cell / Q000097:blood*; D001798:Blood Proteins / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D002846:Chromatography, Affinity / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry / Q000302:isolation & purification*; D006461:Hemolysis; D006801:Humans; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D010949:Plasma / Q000737:chemistry; D040901:Proteomics
chemical_list: D001798:Blood Proteins; D006454:Hemoglobins; D009942:Organometallic Compounds; C088321:nickel nitrilotriacetic acid; D009571:Nitrilotriacetic Acid