Reverse Phase-high-performance Liquid Chromatography (RP-HPLC) Analysis of Globin Chains from Human Erythroid Cells

Abstract

β-hemoglobinopathies are severe genetic disorders characterized either by the abnormal synthesis of the adult β-globin chains of the hemoglobin (Hb) tetramer (βS-globin chains) in sickle cell disease (SCD) or by the reduced β-globin production in β-thalassemia. The identification and quantification of globin chains are crucial for the diagnosis of these diseases and for testing new therapeutic approaches aimed at correcting the β-hemoglobinopathy phenotype. Conventional techniques to detect the different Hb molecules include cellulose-acetate electrophoresis (CEA), capillary electrophoresis (CE), isoelectric focusing (IEF), and cation-exchange-HPLC (CE-HPLC). However, these methods cannot distinguish the different globin chains and precisely determine their relative expression. We have set up a high-resolution and reproducible reverse phase-HPLC (RP-HPLC) to detect and identify the globin chains composing the hemoglobin tetramers based on their different hydrophobic properties. RP-HPLC mobile phases are composed of acetonitrile (ACN) that creates a hydrophobic environment and trifluoroacetic acid (TFA), which breaks the heme group within the Hb tetramers releasing individual globin chains. Hb-containing lysates are loaded onto the AerisTM 3.6-µm WIDEPORE C4 200 Å LC Column and a gradient of increasing hydrophobicity of the mobile phase over time allows globin chain separation. The relative amount of globin chains is measured at a wavelength (λ) of 220 nm. This protocol is designed for evaluating globin chains in (i) red blood cells (RBCs) obtained from human peripheral blood, (ii) RBCs in vitro differentiated from hematopoietic stem/progenitor cells (HSPCs), and (iii) burst-forming unit-erythroid (BFU-E), i.e., erythroid progenitors obtained in vitro from human peripheral blood or in vitro cultured HSPCs. This technique allows to precisely identify the different globin chains and obtain a relative quantification. RP-HPLC can be used to confirm the diagnosis of β-hemoglobinopathies, to evaluate the disease severity and validate novel approaches for the treatment of these diseases.