Accurate Mass Measurement by Electrospray Ionization Quadrupole Mass Spectrometry: Detection of Variants Differing by <6 Da from Normal in Human Hemoglobin Heterozygotes

Mass spectrometry has a basic limitation when human hemoglobin variants are analyzed, because it cannot resolve two globin chains that differ in mass by <6 Da. Several common (beta)-chain variants differ by 1 Da from normal and, hence, when present in heterozygotes, are not resolved from the normal (beta)-chain. Normal and variant chains appear together in the spectrum as a single entity, whose mass is the abundance weighted mean of the two chains. Here we show that such heterozygotes can be detected in 500-fold diluted blood by accurately measuring the mass of the (beta)-chain using an electrospray ionization quadrupole instrument and the (alpha)-chain for internal mass calibration. A statistical analysis of the normal (beta)-chain mass (n = 86) showed that the standard deviation (SD) of the mean was <±0.05 Da (<±3.2 ppm). Hence, at the 95% confidence level (±2 SD), an abnormal (alpha)- or (beta)-chain differing by 1 Da from normal should be detectable in a heterozygote provided its abundance is >10% of total (alpha)- or (beta)-chains, respectively. Variants whose masses lay between 1 and 4 Da from normal were detected in 19 heterozygotes. Moreover, the proportion of each variant estimated from the mass change correlated with the proportion determined by cation-exchange HPLC. Variants were assigned to the (alpha)- or (beta)-chain by combining the sign of the mass change with the polarity change inferred from electrophoretic data. This procedure could be used for screening clinically significant hemoglobin variants.