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.
Division of Clinical Chemistry, Department of Medical Laboratory Sciences and Technology, Huddinge University Hospital C1 74, Karolinska Institutet, SE-141 86 Stockholm, Sweden. dilip.rai@labmed.ki.se
Dilip K
DK
Rai
William J
WJ
Griffiths
Britta
B
Landin
Barbara J
BJ
Wild
Gunvor
G
Alvelius
Brian N
BN
Green
D002138:Calibration; D004586:Electrophoresis; D006455:Hemoglobins, Abnormal / Q000737:chemistry* / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D009154:Mutation / Q000235:genetics; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization
D006455:Hemoglobins, Abnormal