Mass spectral analysis of asymmetric hemoglobin hybrids: Demonstration of Hb FS (alpha(2)gamma beta(s)) in sickle cell disease

Formation of the asymmetric hemoglobin hybrid FS (alpha (2)gamma beta (S)) inhibits hemoglobin S (Hb S) polymerization in vitro and landerlies the pro tective effect of fetal hemoglobin (Hb F) in homozygous sickle cell disease . Conventional methods for separating Hb reveal only symmetric Hb tetramers because of the rapid dissociation of tetramers to dimers relative to the s eparation time for electrophoresis and chromatography. To gain insight into the quantitative distribution of asymmetric Hb FS and other tetrameric spe cies in sickle cell disease, the noncovalent association of Rb subunits in hemolysates was studied by a novel application of electrospray ionization m ass spectrometry (ESI-MS). Mass spectra of both patient and fetal blood rev ealed predominance of tetrameric species with dimer and monomer subunits in lower abundance. ESI-MS analysis revealed the hybrid Hb AF (alpha (2)gamma beta (A)) in hemolysates shown by conventional high-performance liquid chr omatography to contain only the symmetric species Hb A (alpha (2)beta (A)(2 )) and Hb F (alpha (2)gamma (2)). A unique tetramer of average mass 64,558 Da was identified in hemolysates from patients with sickle cell disease in accordance with the calculated mass of the asymmetric Hb hybrid FS. Hybrid Hb species were stable under the ESI-MS conditions employed allowing concur rent determination of the proportions of Hb FS and the symmetrical Hb S (al pha (2)beta (S)(2)). The ratios of Hb FS to Hb S correlated closely (r(2) = 0.96) with those predicted under physiological conditions. (C) 2001 Academ ic Press.