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

Lisa M. Williams, Zongming Fu, Pratima Dulloor, Timothy Yen, Emily Barron-Casella, William Savage, Jennifer E. Van Eyk, James F Casella, Allen D Everett

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)926-930
Number of pages5
JournalProteomics - Clinical Applications
Volume4
Issue number12
DOIs
StatePublished - Dec 2010

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Sickle Cell Anemia
Proteomics
Hemoglobins
Plasmas
Affinity chromatography
L-Selectin
Blood Proteins
Peptides
Biomarkers
Nickel
Affinity Chromatography
Enzyme-Linked Immunosorbent Assay
Apolipoprotein B-100
Plasma (human)
Macroglobulins
Proteins
Mass spectrometers
Hemolysis
nickel nitrilotriacetic acid
Design of experiments

Keywords

  • Biomarkers
  • Hemoglobin depletion
  • Plasma proteomics
  • Sickle Cell disease

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Hemoglobin depletion from plasma : Considerations for proteomic discovery in Sickle Cell disease and other hemolytic processes. / Williams, Lisa M.; Fu, Zongming; Dulloor, Pratima; Yen, Timothy; Barron-Casella, Emily; Savage, William; Van Eyk, Jennifer E.; Casella, James F; Everett, Allen D.

In: Proteomics - Clinical Applications, Vol. 4, No. 12, 12.2010, p. 926-930.

Research output: Contribution to journalArticle

Williams, Lisa M. ; Fu, Zongming ; Dulloor, Pratima ; Yen, Timothy ; Barron-Casella, Emily ; Savage, William ; Van Eyk, Jennifer E. ; Casella, James F ; Everett, Allen D. / Hemoglobin depletion from plasma : Considerations for proteomic discovery in Sickle Cell disease and other hemolytic processes. In: Proteomics - Clinical Applications. 2010 ; Vol. 4, No. 12. pp. 926-930.
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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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T2 - Considerations for proteomic discovery in Sickle Cell disease and other hemolytic processes

AU - Williams, Lisa M.

AU - Fu, Zongming

AU - Dulloor, Pratima

AU - Yen, Timothy

AU - Barron-Casella, Emily

AU - Savage, William

AU - Van Eyk, Jennifer E.

AU - Casella, James F

AU - Everett, Allen D

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N2 - 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.

AB - 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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KW - Sickle Cell disease

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