Hemoglobind™ for Sample Preparation of RBC Proteins for Biomarker Analysis of Stored Blood Products

Hemoglobind™ for Sample Preparation of RBC Proteins for Biomarker Analysis of Stored Blood Products

Sample preparation for in vitro and in vivo discovery of biomarkers from erythrocyte concentrates, platelet concentrates and fresh frozen plasma requires scientists to remove hemoglobin from samples after lysis to obtain accurate results on downstream measurements with minimal hemoglobin interference. Samples containing excess hemoglobin could have hemoglobin-protein interaction resulting in unstable catalytic reactions and produce false positive results for 2DE, DIGE, iTRAQ, SDS-PAGE, MALDI-TOF MS analysis and isotope-coded affinity tags. Thus it is important to standardize strategies for removal of high abundance proteins in proteomic studies of serum/plasma to enhance the detection of low abundance proteins and achieve broader proteome coverage.

The red blood cell has both membrane proteins and cytosolic proteins. For cytosolic protein proteomic studies, it is important to remove or deplete hemoglobin to detect lower abundance proteins. Proteins identified in the cytosolic fraction include hemoglobin, myosin, retinal dehydrogenase, catalase, flavin reductase, serine/threonine-protein phosphatase 2A regulatory subunit B’ isoform 4, bisphosphoglycerate mutase etc. Proteins found in the membrane fraction include ankryrin, arginase, ferritin light chain, glycophorin, ferritin light chain, band 3 anion transport protein, interferon-induced GTP-binding protein, phosphoglucomutase etc. Proper isolation of hemoglobin from the cellular sub-proteome (membrane and cytosolic) could benefit from the Hemoglobind™ protocol. The low abundance and the hydrophobic nature of cell membrane proteins complicate their purification and detection by conventional proteomic studies. So, in order to increase the efficiency of RBC proteome, Hemoglobind™ protocol offers a simple method to remove hemoglobin.

Analysis of membrane and cytosolic proteins based on proteomic techniques for biomarker discovery requires sensitivity and specificity in sample preparation and standardization of sample preparation, storage and pre-analytical steps. Various methods for analysis of RBC proteome are available today such as SDS-PAGE, 2D electrophoresis,MALDI-TOF MS analysis, LC-MS. The peptide ligand library technology have allowed for the exploration of red blood cell proteome by decreasing the erythrocyte dynamic protein concentration range to analyze via the nano-LC-LTQ-Orbitrap MS/MS analysis. Research has shown that patients with certain RBC membrane disorders have decrease in spectrin levels in erythrocyte membrane fractions and such results have increased the effort for RBC proteome biomarker discovery.

Summary of Hemoglobind™ published research:

Hemoglobin depletion strategy is important and in the ‘Biomarker Analysis of Stored Blood Products: Emphasis on Pre-Analytical Issues’, Int. J. Mol. Sci. 2010, 11, 4601-4617; article authors cite the HemogloBind™ reagent method as ideal for hemoglobin capture and depletion, followed by labeling-based quantitation and LC-MS/MS identification. Such research has led to the development of establishment of interactome and network maps. HemogloBind™ is engineered for a high degree of selectivity and does not cross react with most common serum components, making it an excellent tool in numerous applications.

Biotech Support Group also has HemoVoid™ which depletes hemoglobin from red cell lysates while improving the resolution of less abundant blood proteins. Hemoglobin voids in flow-through and this allows for the analysis of hemoglobin variant and hemoglobin binding proteins. The eluted proteins contain all the RBC proteins minus the hemoglobin available for biomarker studies.

For more information: http://biotechsupportgroup.com/hemovoid-hemoglobin-depletion-reagent

Visit the Biotech Support Group website: http://biotechsupportgroup.com/hemoglobind-hemoglobin-removal-and-capture

References for Hemoglobind ™ & Hemovoid ™:

• Functional 20S proteasomes in mature human red blood cells Sudha Neelam¹, David G Kakhniashvili¹, Stephan Wilkens¹,Stephen D Levene² and Steven R Goodman1 Exp. Biol. Med. 2011;236:580-591
• Kyoungsook Park, Christopher D. Saudek, and Gerald W. Hart (2010) Increased Expression of β-N-Acetylglucosamindase (O-GlcNAcase) in Erythrocytes from Prediabetic and Diabetic Individuals. Diabetes. 59(7):1845-50.
• Delobel J., Rubin O., Prudent M., Crettaz D., Tissot J.-D., Lion N.(2010) Biomarker Analysis of Stored Blood Products: Emphasis on Pre-Analytical Issues. International Journal of Molecular Sciences. 11(11):4601-4617
• Alvarez-Llamas, G., de la Cuesta, F., Barderas, M. G., Darde, V. M., Zubiri, I., Caramelo, C. and Vivanco, F. (2009), A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE. ELECTROPHORESIS, 30: 4095–4108
• Zihao Wang, Kyoungsook Park, Frank Comer1, Linda C. Hsieh-Wilson, Christopher D. Saudek, and Gerald W. Hart (2008) Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes Mellitus. Diabetes 58, 309-317.
• Sarawathi,et al., Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry, Biochim Biophys Acta. 1999 Feb2; 1426(3):483-90.

Suggested References:

• Rubin, O.; Crettaz, D.; Tissot, J.D.; Lion, N. Pre-analytical and methodological challenges in red blood cell microparticle proteomics. Talanta 2010, 82, 1–8.
• Maione TE, Gray GS, Hunt AJ, Sharpe RJ. Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity. Cancer Res. 1991;51:2077-2083
• Scher W, Holland JG, Friend C. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro. I. Partial purification and identification of hemoglobins. Blood 1971; 37: 428-437