Hemoglobind ™ is an Ideal sample Preparation Reagent to Develop the Most Complete and Informative Human RBC Proteome Experimental Data

Hemoglobind ™ is an ideal sample preparation reagent to develop the most complete and informative human RBC proteome experimental data

Biotech Support Group’s goal is to allow scientists to develop the most complete and informative human RBC proteome experimental data possible by combining high-accuracy, high-sensitivity protein identification technology with Hemoglobind™ for sample preparation. Approximately, 98% of the protein composition of a red blood cell(RBC) is hemoglobin (Hb), the oxygen-carrying protein of erythrocytes. Hemoglobind™ has numerous applications as a sample preparation reagent for hemoglobin protein analysis.

Analysis of the cytoplasmic RBC proteome is challenging because hemoglobin is a heavily abundant protein that masks other proteins. This is evident by the total hemolysate which yields a hemoglobin spot on 2D gel.After implementing the Hemoglobind™ protocol, smearing spots of other proteins are detectable as spots on 2D gel because Hemoglobind™ binds to hemoglobin with a high degree of specificity. Pretreatment of serum or plasma samples from human, mouse, sheep, bovine, goat, rat, and calf with Hemoglobind™ as a prefractionation technique for purification and/or analysis of hemoglobin has many benefits over size exclusion chromatography, cation exchange chromatography, in-solution isoelectrofocusing. Hemoglobind is compatible for highthroughput applications and has a flexible loading capacity. Moreover it does not capture proteins other than hemoglobin thus making it ideal for applications in blood substitutes, enzyme recovery and analytical interferences.

Two published articles highlight the application of Hemoglobind for hemoglobin depletion from RBC lysates.

Increased Expression of β-N-Acetylglucosamindase (O-GlcNAcase) in Erythrocytes from Prediabetic and Diabetic Individuals.

Erythrocyte proteins are highly O-GlcNAcylated. In individual with pre-diabetes and diabetes, the level of O-GlcNAcase expressed significantly increases.

From serum samples, erythrocyte proteins were extracted and hemoglobin was depleted followed by sonication and centrifugation. From the red blood cell lysates hemoglobin is efficiently depleted using HemogloBind from Biotech Support Group. Because HemogloBind™ is engineered for a high degree of selectivity and does not cross react with most common serum components, subsequent analysis of O-GlcNAcylation process in erythrocyte proteins is done by Western blotting using an O-GlcNAc specific antibody. Finally, the study of O-GlcNAcase allows for developing, validating, and qualifying biological markers that are compared with the level of A1C.

Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes Mellitus.

O-GlcNAc actively cycles on erythrocyte, regulates insulin signaling and is a mediator of glucose toxicity. Therefore studying it may reveal potential biomarker for diagnoses of diabetes. Highly efficient enrichment methods based Hemoglobind overcome the challenges of low stoichiometry, suppressed ionization efficiency in presence of unmodified peptides, and intrinsic lability in gas phase mass spectrometric methods. In this paper, authors used Hemoglobind to study erythrocyte proteins and compared it with their abundance between normal and diabetic samples proteins. Blood samples were obtained from normal and diabetic patients collected into a vial containing EDTA and OGlcNAcase inhibitor PUGNAc. Next the researchers fractionated the blood cells to isolate erythrocytes. After erythrocytes were lysed and centrifuged, the supernatant containing hemoglobin was partially depleted by HemogloBind from Biotech Support Group

Published research has shown that the RBC cytosol contains proteins relating to diseases such as myeloproliferative disorders or malignancies of precursor hematopoietic cells. Thus, HemogloBind™ is the sample reagent of choice for studying the RBC proteome data and for clinical applications in bilirubin analysis and bulk serum clarification.

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For more published references on Hemoglobind™:

Baion, C.M. & Ali, A.C., Evaluation Of HemogloBind™ For Removal Of O-Raffinose Crosslinked Hemoglobin (Hemolink™) From Serum, poster AACC Meeting 1997.

Padilla, S., Convenient Method for Decreasing the Amount of Hemoglobin in Tissue Samples Without Affecting the Level of Cholinesterase Activity, unpublished personal correspondence, 1994.

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

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.

References

• 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.
• 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.
• Wright C, Bergstrom D, Dai H, Marton M, Morris M, et al. (2008) Characterization of globin RNA interference in gene expression profiling of whole-blood samples. Clin Chem 54: 396–405.
• Bhattacharya, D., Mukhopadhyay, D. and Chakrabarti, A. (2007), Hemoglobin depletion from red blood cell cytosol reveals new proteins in 2-D gel-based proteomics study. PROTEOMICS – Clinical Applications
• 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. J Proteome Res. 2005;4: 748-757.
• Kakhniashvili DG, Bulla LA Jr, Goodman SR. The human erythrocyte proteome: analysis by ion trap mass spectrometry. Mol Cell Proteomics. 2004;3: 501-509