PubMed · 8779434
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C H Shackleton, H E Witkowska. 1996-01-01. Characterizing abnormal hemoglobin by MS.. https://doi.org/10.1021/ac9618031
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The mass spectrometric techniques have been used for the detection, characterization and quantification of variant hemoglobins (Hbs). Electrospray ionization mass spectrometry (ESIMS) technique allows the analysis of mixtures of intact globins by giving accurate molecular weights and structural informations. By the application of ESIMS method, we found 18 kinds (25 cases) of variant Hbs including 2 new variants, Hb Sagami [beta 139 Asn-->Thr] and Hokusetsu [beta 52Asp-->Gly] and characterized amino acid substitutions and post-translational modifications of Hbs for past 5 years. In the present study, we applied current advanced MS techniques such as nanospray MS (nanoESIMS), a ultra-high sensitive analysis and in-source fragmentation (MSMSMS; MS3) technique. High sensitivities in nanospray ESIMS are achieved by utilizing an integrated C18 column nanoscale ES tip which can directly connect to gradient LC separations at flow rates between 0.5 to 2.0 microliters/min. We handled an immobilized trypsin (Poroszyme) and nanoESIMS and MSMS techniques to determine the amino acid substitutions of variant Hbs. The globin mixtures were rapidly cleaved by Poroszyme and analyzed by nanoESIMS and MSMS. We also operated in-source fragmentation (MSMSMS; MS3) technique using a proline fragment or [M + 14H]14+ of intact normal and variant beta globin and confirmed the sequence of amino acids of variant Hbs. These techniques could be used extensively for rapid characterization of variant Hbs as well as the other proteins in body fluids at very high sensitivity.
Abnormal hemoglobin is one of the most frequent genetic disorder, of which about 700 variants have been reported in the world. Some abnormal hemoglobins such as HbS and HbC are endemic among limited human populations. We have found five subjects with abnormal hemoglobin in routine hematology analysis using an automated system Sysmex NE-7000. We isolated genomic DNA from the peripheral blood leukocyte of each subject, and amplified a 558 bp fragment of DNA including the entire exons 1 and 2 as well as intron 1 of the beta-globin gene. The amplified DNA fragments were subjected to direct sequencing by the dideoxy termination method on an automated DNA sequencer. The sequence analysis showed the abnormalities including HbA/C, HbS/C, HbE/E, and HbA/G-Szuhu. In the present study, we report a simple method for the mutual differentiation of these abnormal hemoglobins using restriction fragment length polymorphism (PCR-RFLP) method. Digestion of the amplified fragments described above with a restriction enzyme MnlI gave different RFLP patterns for HbA, HbC or S, HbE, and for HbG-Szuhu. RFLP using another enzyme, DdeI, could distinguish for HbS from HbC.
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