Diagnosis of Hemoglobinopathy and β-Thalassemia by 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry of Hemoglobin from Blood

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Abstract
Background: Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. Methods: We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21-Tesla Fourier transform ion cyclotron resonance–tandem mass spectrometry (21-T FT-ICR MS) for hemoglobin (Hb) variant de novo sequencing and β-thalassemia diagnosis. Results: We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and β subunits (δ/β) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose β-thalassemia trait caused by a mutation in 1 HBB gene. Conclusions: We found 21-T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The developed method has the potential to be translated to lower resolving power mass spectrometer (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and β-thalassemia diagnosis (MS1).

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