Seung Jun Lee (Presenter)
Seoul National University Hospital
Bio: Clinical Fellow, Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea
Authorship: Seung Jun Lee (1,2), Kyunghoon Lee (1,2), Sun-Hee Jun (3), Sang Hoon Song (1,2), Kyoung Un Park (1,3), Junghan Song (1,3)
(1) Department of Laboratory Medicine, SNUCM, Seoul, Korea (2) Department of Laboratory Medicine, SNUH, Seoul, Korea (3) Department of Laboratory Medicine, SNUBH, Seongnam, Korea
| Short Abstract We developed an UPLC-MS/MS method to identify hemoglobin (Hb) G-Coushatta and Hb Yamagata, common Hb variants in Korea, among suspected specimens detected by conventional HbA1c analyzers. All Hb variant specimens and normal controls were tested in UPLC-MS/MS system after the treatment with endoproteinase Glu-C. Two peaks, 689.8 m/z [M+2H+] and 832.4 m/z [M+3H+] at 6.36 min, and one peak, 832.4 m/z [M+3H+] at 10.78 min, were found to distinguish Hb G-Coushatta and Hb Yamagata from other Hb variants and normal, respectively. And the zygosity for these variants was determined by the intensity of two peaks: 657.8 m/z and 460.2 m/z. |
Long Abstract
BACKGROUND
More than 1,000 hemoglobin (Hb) variants are currently known. Hb G-Coushatta, Hb Yamagata, Hb Chad, and Hb Queens are common in Korea although the commonly occurring hemoglobin variants are HbS, HbC, HbE, and HbD in other countries. Here, we developed an ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method to identify Hb G-Coushatta and Hb Yamagata among suspected specimens detected by conventional HbA1c analyzers.
METHODS
Red blood cells were separated, washed and hemolysed in distilled water. The hemolysate was treated with endoproteinase Glu-C and kept in storage at -70¡É. A Shimadzu Nexera X2 UPLC and AB SCIEX Triple QuadTM 6500 were used for UPLC-MS/MS analysis. A Jupiter Proteo column (2.0 ¡¿ 50 mm, 4 ¥ìm) and gradients of water and acetonitrile in 0.1% formic acid were used for UPLC separation after this sample preparation. Total running time was 20 min and flow rate was 300 ¥ìL/min. On the basis of the protein sequence, we calculated molecular weight and mass to charge ratio of possible protein sequences for each variants: Hb G-Coushatta, Hb Yamagata, Hb Chad, and Hb Queens to compare with peaks obtained by UPLC-MS/MS.
RESULTS
All Hb variant specimens confirmed by DNA sequencing method and two normal controls were carried out in UPLC-MS/MS system. Two peaks which consist with calculated 689.8 m/z [M+2H+] and 832.4 m/z [M+3H+] were found at 6.36 min to identify Hb G-Coushatta and one peak, 832.4 m/z [M+3H+], was found at 10.78 min to identify Hb Yamagata. It was found that all Hb G-Coushatta cases were heterozytes because the intensity of 657.8 m/z [M+2H+] peak at 6.34 min was about half of peak intensity observed in normal control. For Hb Yamagata, one peak, 832.4 m/z [M+3H+], was found at 10.78 min to be distinguish from normal Hb and other variants. And all cases were heterozygotes due to comparison with the peak intensities, 460.2 m/z [M+3H+], of Hb Yamagata and normal control. However, there was no peak to identify Hb Chad or Hb Queens.
CONCLUSIONS
We distinguished Hb G-Coushatta and Hb Yamagata from other Hb variant and normal Hb using UPLC-MS/MS. Our method should be useful to identify these variants rapidly without using molecular methods.
Key words: hemoglobin variant, mass spectrometry, hemoglobin G-Coushatta, hemoglobin Yamagata
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