Analytical
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Hb Quebec-Chori discovered The most widely known hemoglobin mutation is sickle-cell anemia, a relatively common condition among people of African and Middle Eastern ancestry. In sickle cell anemia, normally biconcave red cells become elongated on deoxygenation because of a single mutation in the hemoglobin P-chain that drastically lowers the solubility of deoxy-Hb S and triggers its polymerization. Polymerized deoxy-Hb S forms long bundles of multistranded threads, forcing the red cells into the distinctive rigid sickle shape. The deformed cells block vascular capillaries and cause multiple organ damage, morbidity, and early mortality. A recent case study illustrates the power of MS to diagnose a previously unreported variety of sickle cell disease and suggests that the technique may be useful for other hemoglobinbased analyses. The study involved a patient who apparently had the sickle cell trait, as established by her isoelectric focusing pattern, which showed the presence of Hb A and Hb S (see Figure 1, lane 7). Although the patient displayed many symptoms of the disease, we did not understand why sickling was occurring, because the ratio of Hb S to Hb A was not large enough to trigger polymer formation.
hemoglobinopathy diagnostics, technological improvements are of utmost importance because they alone can secure the economic feasibility of the procedure. Automation is one obvious way of decreasing instrument and analyst time. In addition, highly accurate peptide mapping using MALDI/TOFMS with reflectron detection offers the analyst the possibility of easily screening proteolytic peptide mixtures for abnormal fragments. Samples could be processed by LC/MS/MS or other MS sequencing techniques, including postsource decay in MALDI/TOFMS, only when peptide sequencing was required. A "utopian dream" approach to variant protein analysis by MS would be sequencing an intact protein molecule by MS/ 32 A
One possibility was the presence of another mutation somewhere in the hemoglobin molecule that encouraged deoxy-Hb S to polymerize and form the sickle-shaped cells. Although electrophoretic methods are useful screening procedures for detecting abnormal hemoglobin variants, the lack of separation where a mutation is presumed to exist poses a serious limitation: When the net charge of the molecule is negligible, the mutation is electrophoretically "silent." Such electrophoretically silent mutations are likely to occur when one neutral amino acid is replaced by another or when acidic or basic amino acids are replaced by their counterparts. In this particular case, the putative "new" hemoglobin electrophoretically migrated to the position of either A or S to give a false sickle cell trait pattern. Because we thought that the mutation would introduce a difference in globin molecular mass, we used ESIMS to characterize the globins. The ESI mass spectrum of globins obtained from the patient showed a-globin (15126 Da), ps-globin (15837 Da), and an additional (3-globin variant (15879 Da). Normal P-globin (15867 Da) was absent from the globin sample. This patient clearly had a combina-
MS. The groundwork for intact globin fragmentation studies was laid by Smith's group (19), who analyzed a series of P-chain mutants. Later it was shown that a globin exhibits differing fragmentation pathways depending on the charge state of the precursor ion (20). Recently, in collaboration with Green and Morris, we successfully applied fragmentation of an intact variant oc-chain to characterize the mutation in an unknown sample (21). The results suggest that intact globin fragmentation could be used to confirm certain common mutations that reside within the well-mapped regions. One of the most difficult tasks for the future is a definitive diagnosis of the absence of hemoglobinopathy, which often is required in evaluating a clinically signifi-
Analytical Chemistry News & Features, January 1, 1996
tion of hemoglobinopathies—she inherited the 15879-Da p-variant from her mother, who carries the trait, and the Ps-globin from her father. The 15879-Da P-variant combines with the a-globin to form a hemoglobin indistinguishable from Hb A in electrophoretic assays. From the HPLC pattern of the tryptic digest, we identified PT10 as the fragment containing the mutation, introducing a 12-Da increment in molecular mass. We deduced that a Thr -> He replacement had occurred. Considering only molecular mass, leucine also could have been the new amino acid; however, no single mutation in the codon used for threonine in mis peptide could produce the resulting amino acid. The major problem remaining was that pTIO contained two threonines, at positions 84 and 87. When the peptide was analyzed by MS/MS with an array detector, we concluded that the fragment ions in the product-ion spectrum could only have arisen if the mutation occurred at position 87, and the presence of a wl2 fragment ion typical of an isoleucine side chain was observed (22). The new hemoglobin was called Hb Quebec-Chori after the Canadian province in which the patient lived and the institute at which the hemoglobin was characterized (23).
cant case. There currently is no consensus regarding the type of tests needed for a negative diagnosis. Although ESIMS offers the possibility of screening for hemoglobin mutations independent of net charge and hydrophobicity of globin, the existence of silent mutations cannot be definitively dismissed. A fast sequencemapping approach that can quickly and inexpensively examine 100% of the protein structure is needed for such a negative diagnosis. We appreciate and highly value all support, assistance, and encouragement that we have received from our colleagues at Children's Hospital Oakland and other institutions. We also thank Terri Brown for her valuable and skillful help in preparing the manuscript. This work was supported by National Institutes of Health Sickle Cell Center Grant HL20985.
