Electrospray Ionization Mass Spectrometric Analysis of the Globin Chains in Hemoglobin Heterozygotes Can Detect the Variants HbC, D, and E

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Abstract
While we agree that ESI-MS cannot detect the zero mass change mutations (Lys↔Gln and Leu→Ile), we maintain it is not necessary to resolve the variant and normal globin chains in heterozygotes to detect variants that differ in mass from normal by ±1 Da (Glu↔Lys, Glu↔Gln, Asp↔Asn, Asn→Ile). In 2003, Rai et al. (3) showed that variants differing by 1 Da from normal can be detected if present at >10% abundance. In that report, the normal β-chain mass was determined with a precision of 0.05 Da SD, which resulted in a 0.10 Da mass change being detectable with 95% confidence. We routinely analyze Hb on a quadrupole instrument and, owing to improved performance since 2003, generally achieve ≤0.03 Da SD on the normal β-chain when using the α-chain for internal calibration. For example, 50 normal blood samples analyzed over the last 4 months gave a mean β-chain mass of 15 867.255 Da (0.026 Da SD). On 22 D-Punjab (β121Glu→Gln) heterozygotes, the mean β-chain mass was 15 866.832 Da (0.039 Da SD), which is 0.423 Da lower than that determined for the normal β-chain. This mass difference is readily detected and even allows the mean abundance of the variant β-chain to be estimated as 43.0%, which agrees with the mean abundance determined from the HPLC data associated with each sample (41.0%, 3.2% SD, n = 22). The above SD values imply that mass changes down to between 0.1 and 0.2 Da can be reliably detected. A −0.2 Da mass change would suggest a heterozygous −1 Da mutation (Glu→Lys, Glu→Gln, Asp→Asn, or Asn→Ile) at 20% abundance or heterozygous Lepore-Boston-Washington (normal β − 2 Da) at 10% abundance.