Determination of neutral haemoglobin variants by immobilized pH gradient, reversed-phase high-performance liquid chromatography and fast-atom bombardment mass spectrometry: The case of a Hb torino α 43 (CE1) Phe→Val

A neutral haemoglobin variant was identified by the combined use of different analytical methods. Isoelectric focusing on immobilized pH gradients (under denaturing conditions) allowed to detect and a. chain variant. HPLC of tryptic digest showed that its aminoacid variation resided on Ta6 peptide (position 41—56). In the fast-atom bombardment (FAB) positive mass spectrum of the tryptic digest, the protonated molecular ion of the Ta6 peptide occurred 48 mass units lower than the normal Ta6 fragment, corresponding to a Phe -> Val substitution. To a partial sequence determination (aminopeptidase digestion of the Ta6 peptide, followed by amino acid determination and FAB-MS analysis of the digestion-generated mixture) the substitution appeared to be on Phe43 (CE1). This variant is already known as Hb Torino. The procedure here described proved to be fast and simple, and feasible whenever neutral variants are supposed to occur.