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.
Rai
Dilip K
DK
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
Griffiths
William J
WJ
Landin
Britta
B
Wild
Barbara J
BJ
Alvelius
Gunvor
G
Green
Brian N
BN
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