HemoVoid™ For Validation of Red Blood Cell Lysate Proteome And Analysis Utilizing 2D-PAGE and Orbitrap MS

The identification of proteins in erythrocytes for proteomics studies is hampered by the huge abundance of hemoglobin (Hb) which hides other proteins and makes detection in 2-D gel-based separation difficult. It is therefore important to deplete hemoglobin and enrich low abundance proteins. Biotech Support Group's HemoVoid™ is cited in a paper by authors Walpurgis et al titled, "Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D-PAGE and Orbitrap MS" in the journal Electrophoresis. Authors Walpurgis et al demonstrate HemoVoid™'s application in the development of a protocol for proteomic analysis of hemoglobin-depleted RBC lysates in human blood from healthy donors. After using HemoVoid™, 2D-PAGE comparison of the unextracted hemolysate and the HemoVoid™ depleted hemolysate displays disappearance of the prominent and smeared hemoglobin 'spot' formerly containing high amount of hemoglobin on the gel is significantly reduced. Thus HemoVoid™ allows scientists to detect and study non-hemoglobin proteins using subsequent LC-MS/MS analysis for study of cytosolic proteome. This article describes the HemoVoid™ process as a "…very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample", and that "…a two-dimensional reference map (pH 4-7) of the cytosolic red blood cell proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution two dimensional protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic red blood cell proteome." Authors recorded that HemoVoid™ removed more than 98% of cellular hemoglobin, indirectly concentrated minor proteins and eliminated unbound hemoglobin. Upon comparison of the untreated and HemoVoid™ treated RBC lysates in the flowthrough and wash fractions by SDS-PAGE, they discovered the untreated RBC lysate showed two intense hemoglobin-derived bands (approximately 15 and 30 kDa), and the HemoVoid™-treated RBC lysate did show several bands which were not visible prior to hemoglobin depletion possibly representing non-hemoglobin proteins. Flow-through and three wash fractions contained some bands plus hemoglobin bands. Significant increase in the number of detected bands after hemoglobin depletion and therefore enormous gain of information is observed plus some observed protein losses accompanying the HemoVoid™-based sample preparation. HemoVoid™, a hemoglobin depletion reagent kit, removes hemoglobin from whole blood, hemolyzed serum and erythrocyte cell lysates for proteomics study while concentrating low abundance proteins. The HemoVoid™ protocol uses mild buffers preserving native enzyme activity of samples in elution fractions. Mild elution maintains tertiary structure and simple transfer to secondary analysis. Currently, researchers are using HemoVoid™ for a hemoglobin enrichment protocol from blood samples for hemoglobin variant research (HbS, HbF, HbA, HbA1c, Thalassemia, etc.). Our unique surface technology approach, while not based on antibodies or peptide combinations, is nevertheless highly selective and efficient for enrichment of low abundance proteins, with the advantages of a consumable and cost-effective product. References Katja Walpurgis, Maxie Kohler, Andreas Thomas et al.Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D-PAGE and Orbitrap MS.Electrophoresis.2012; Mizukawa, B., George, A., Pushkaran, S. et al. Cooperating G6PD mutations associated with severe neonatal hyperbilirubinemia and cholestasis.Pediatric Blood Cancer.2011;56: 840-842. Sudha Neelam, David G Kakhniashvili, Stephan Wilkens et al. Functional 20S proteasomes in mature human red blood cells Experimental Biology and Medicine.2011;236:580-591 For more information on HemoVoid™ and references click here. Suggested References Tyan Y-C, Jong S-B, Liao J-D, et al. Proteomic profiling of erythrocyte proteins by proteolytic digestion chip and identification using two-dimensional electrospray ionization tandem mass spectrometry. Journal of Proteome Research. 2005;4: 748-757 Kakhniashvili DG, Bulla LA Jr, Goodman SR. The human erythrocyte proteome: analysis by ion trap mass spectrometry. Molecular Cellular Proteomics.2004;3: 501-509. Low TY, Seow TK, Chung MCM. Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry.Proteomics. 2002;2: 1229-1239.

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