Hemoglobin depletion from plasma: considerations for proteomic discovery in sickle cell disease and other hemolytic processes

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, -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.

Trial registration: ClinicalTrials.gov NCT00072761.

Conflict of interest statement

The authors have declared no conflict of interest.

Figures

Figure 1

Coomassie-stained 1D SDS-PAGE of plasma proteins bound to Ni-NTA from individuals with sickle cell disease and hemolysis or normal control. High= increased hemolysis (average reticulocyte count 16%; average bilirubin 7.6 mg/dL); low = low hemolysis (avg. reticulocyte count 4.6%; average bilirubin 2.4 mg/dL); N= no hemolysis. Molecular weight markers (kDa) are indicated on left side and representative bands isolated for mass spectrometry identifications indicated on the right.

Figure 2

Ni-NTA decreased plasma concentrations of TSP1 (A) and L-selectin (B). A. Average plasma TSP1concentrations before and after depletion were 20.8 and 10.6 μg/mL for patients with high hemolysis (n=3), 12.8 and 8.3 μg/mL for low hemolysis (n=3) and 4.0 and 2.3 μg/mL for no hemolysis (n=2), respectively. B. Average Plasma L-selectin concentrations before and after depletion were 1.7 and 1.0 μg/mL for patients with high hemolysis (n=3), 1.4 and 1.1 μg/mL for low hemolysis (n=3) and 1.2 and 1.1 μg/mL for no hemolysis, respectively (n=2).