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Characterizing Abnormal Hemoglobin by MS Structural confirmation of hemoglobin variants can lead to early diagnosis of hemoglobin-based illnesses
H
emoglobins with abnormal structure can occur as a result of random mutations or as a defense mechanism. For example, populations exposed to pandemic malaria successfully developed various red blood cell defects that conferred resistance to the disease (1). Many antimalaria mechanisms involve mutations that affect hemoglobin-globin structure, level of expression, and developmental regulation; if the well-being of a carrier is not significantly undermined in the process, the hemoglobin mutation is passed on as a unique survival tool. Sometimes, however, acute red cell alteration and/or accumulation of the modifying elements in an individual results in morbidity and premature death. It is estimated that 150 million people worldwide carry hemoglobin variants, and a small proportion of these people are symptomatic. Until recently, the presence of clinical symptoms was the most likely reason for undertaking hemoglobin char-
C e d r i c H. L. S h a c k l e t o n a n d H. E w a W i t k o w s k a Children's Hospital Oakland Research Institute 0003-2700/96/0368-29A/$12.00/0 © 1995 American Chemical Society
acterization. However, because in almost 40 states there have been increases in mandatory screening of newborns for hemoglobin-based illnesses, calls for analysis of uncommon variants are more frequent. Although the primary goal of newborn screening is to detect hemoglobinbased illnesses (and thus provide early medical intervention), screenings also detect uncommon variants with unknown clinical significance. Given limited financial resources, it is impossible to characterize all variants, and structures are usually confirmed only when medically necessary. However, besides academic interest, conclusive diagnosis of all detected hemoglobin variants is of practical value. In a small number of cases, it would be cost-effective to provide early warning and counseling regarding uncommon variants that aggravate clinical symptoms when co-inherited with common hemoglobin variants. In
most situations, parents and health-care providers would be assured that the child faces little risk from the presence of an exotic abnormal hemoglobin. Molecular biology and MS will be used extensively in routine hemoglobin diagnostics in the near future. Although restriction enzyme-based techniques are ideal as confirmatory tests in a targeted analysis, MS is better suited for evaluating the unknowns. In this Report we discuss the characterization of hemoglobin and its variants by MS and demonstrate the potential uses of the technique in the clinical setting. Hemoglobin structure and its variations
Adult hemoglobin (Hb A) is a tetramer of two oc-globin and two |3-globin chains, each carrying a heme moiety. The subtle conformational changes between the oxy("relaxed" or "R") and deoxy- ("tense"
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or "T") forms of hemoglobin allow unique cooperation between hemoglobin subunits: Oxygen binding to one subunit increases the capacity for oxygen at other subunits in the tetramer. Originally, hemoglobins were named alphabetically in the order in which they were detected on electrophoresis gels as bands of different mobility. However, given the high rate of discovery of new variants, the alphabetic system was dropped in favor of naming hemoglobins after the geographical locations where they were first encountered or described. More than 600 mutant hemoglobins have been identified to date, and a list of known variants is updated annually by the International Hemoglobin Information Center in Atlanta, GA. If we consider only mutations with one error in globin genes (one-point mutations), 1690 different aand p-globins are possible; however, some are undoubtedly incompatible with life (2). Although single amino acid substitution causes ~ 95% of globin structural abnormalities, chain elongation and deletion, reading frame shift, and gene fusion can also cause problems. Structural changes to the surface amino acid residues are usually innocuous, but sickle cell mutation is an obvious exception. Internal mutations are often associated with significant alteration in hemoglobin function.
inates electropherograms. The classic approach to variant hemoglobin characterization starts with isolation of the abnormal protein followed by tryptic digestion (4). Most workers use a modified version of the HPLC method, designed by Rahbar et al. (5), for tryptic peptide separation even though small hydrophilic di-, tri-, and tetrapeptides are problematic when using this technology. With experience, such HPLC tryptic maps become straightforward to interpret because the pattern is consistent and the absence of a known peptide or presence of a new peak alerts one to the peptide containing the mutation. Often only the aberrant peak needs to be mass analyzed, and this analysis alone may give the information required for final characterization. In some cases an accurately determined mass differential between a normal tryptic peptide and the corresponding variant peptide gives an unambiguous answer as to which amino acid has changed, but sequencing the peptide is frequently necessary. The use of MS to characterize hemoglobin dates back to the pioneering study
of Matsuo and colleagues in 1981 (6). Initially, they used field desorption MS to obtain mass spectral fingerprints of the peptides formed by trypsin digestion of a-, P-, f, and 8-hemoglobin chains and showed an expected mass difference of -30 Da for the sickle cell hemoglobin peptide. During subsequent years, the Osaka group (7) characterized many a-, P-, f, and 5-hemoglobin mutants using fast atom bombardment (FAB) MS. Several other groups also conducted pioneering studies in the mid-1980s (4,8). The tryptic fragments from hemoglobin or separated globin chains can also be analyzed by FAB without separation (9, 10). Whereas most of the peptides are detected, the signals have variable intensity, as expected for a mixture of species with different propensity for sputtering under FAB conditions. In favorable circumstances, hemoglobin mutations can be localized with minimal sample preparation; however, in most cases, fractionation of peptides before analysis is more productive. When characterizing mutations that introduce only minute differences in molecu-
Interestingly, twice as many structural mutations have been described for 13-globin as for a-globin. Close to 100 variants have been characterized using MS as the primary analytical technique, at least 12 of which are variants described for the first time (3). As in all inborn errors, the rare mistakes of nature in hemoglobin production have provided the most knowledge of its structure. In addition to the clinical importance of studying new hemoglobin variants with potential harmful effects, many new variants contribute to our knowledge of hemoglobin assembly, thus making their study worthwhile. Characterizing variant hemoglobin For many years electrophoretic methods have been used to establish the normality of an individual's hemoglobin (Figure 1). In normal adults, only the Hb A band dom30 A
Figure 1 . Hemoglobin isoelectric focusing on agarose slab gel in a pH 6 - 8 gradient. Hemoglobins found in a normal individual (A), a carrier of sickle cell trait (A and S), and a patient with sickle cell disease (S) are shown in lanes 2, 3, and 5, respectively. Hemoglobin patterns of a carrier of Quebec-Chori with A and a patient with Quebec-Chori and S are shown in lanes 6 and 7, respectively. Lanes 1, 4, and 8 contain control mixtures of hemoglobins C, E, S, D, F, and A.
Analytical Chemistry News & Features, January 1, 1996
lar mass, high accuracy in mass measurement is required. (Ironically, mutations of this type are fairly common.) Prome et al. (11) addressed this problem by using tandem MS for variant hemoglobin characterization. Four-sector mass spectrometer systems with array detectors can handle all sequencing problems, including distinguishing between amino acid residues with identical mass (e.g., He and Leu). Current practices The introduction of electrospray ionization (ESI) MS allowed intact proteins to be mass analyzed for the first time. Human globins are dream molecules for ESIMS because of their favorable size (~ 15-16 kDa), an adequate number of protonation sites, the distinct mass differences among different types of globins, and the absence of significant post-translational modifications. An accuracy of 0.01% is achievable for each globin component when physiological mixtures are analyzed. The initial work on normal adult and fetal hemoglobins was done by Green et al. (12), and the technique was used widely for variant characterization soon afterward. For practical purposes, 25% baseline separation for two globins that differ in mass by 12 Da is close to the maximum that can be practically achieved in ESI using a quadrupole detector and without resorting to mathematical deconvolution. Performing mathematical deconvolution using maximum entropy software brings this minimum measurable molecular mass difference down to 6 Da for proteins in the 15- to 16-kDa range (13). Using a separation step before ESIMS offers the analyst a practical remedy for problems caused by limitations in mass resolution. When reversed-phase LC is interfaced with ESIMS, masses of all resolved globins can be accurately measured and 90% of the material can be collected for further analysis (14). When the LC/MS approach is used, putative variants of low abundance gain credibility. For example, there is no confusion about whether a small peak with a molecular mass 21 Da higher than normal is a globin variant or a sodium salt of its normal counterpart. It is particularly beneficial to combine separation and ESIMS in the analysis of
Figure 2. LC/ESI mass spectrum of a mixture of underivatized peptides from a carrier of Hb Hasharon. Reconstructed single-ion chromatograms for normal (a) and abnormal (b) aT6 peptides. (c) Base peak intensity LC/MS profile. The abnormal peptide ocT6 (arrow), which elutes with the normally present pT2, is characteristic of Hb Hasharon.
proteolytic peptides. The original hemoglobin tryptic peptide MS and MS/MS studies by Covey and colleagues were done on-line with HPLC (15). Tryptic fragments of hemoglobin give primarily double-charged molecular ions, although more highly charged peptides are observed with increased length. Our routine strategy uses the laborious separation of abnormal hemoglobins (or globins) only when the mass difference between the normal and abnormal counterpart is 1 Da, when a variant is present at a relatively low level, or when a significant amount of fetal hemoglobin is present in addition to the adult hemoglobin. Otherwise, the mixture of globins is digested and analyzed by LC/MS, as shown in Figure 2. State-of-the-art hemoglobin variant analysis by HPLC/ESIMS/MS was demonstrated by Terry Lee of the Beckman Research Institute of the City of Hope, who combined the sensitivity and resolution of capillary HPLC with efficient collision-induced dissociation and versatile software, allowing the rapid switch from MS to MS/MS after detecting a relevant peak. Matrix-assisted laser desorption/ ionization (MALDI) shows great promise for protein and peptide characterization.
Although currently available resolution discourages the use of MALDI/time-offlight MS (TOFMS) to detect mutations in intact globins, crude proteolytic digest mixtures can be analyzed successfully, and unequivocal detection of abnormal peptide can be achieved. For example, Biemann's group (16) successfully used MALDI MS to examine proteolytic digest mixtures of normal and cross-linked human p-globin. Historically, electrophoresis and isoelectric focusing have been central to hemoglobin analysis, and integration of these techniques with MS would bridge the gap between old and new technologies. Both methods provide enough material (at least for the major bands) to allow full mass spectral analysis, and ESIMS of two bands eluted from an isoelectric focusing gel of a sample from a patient with sickle cell trait yields excellent spectra of Hb A and Hb S (4). In another example of such hyphenated methods, preparative isoelectric focusing in narrow pH gradients followed by MS has been used to characterize hemoglobin variants (17,18). Prospectus Although funding concerns may redefine the directions that MS takes in the field of
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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.
References (1) Nagel, R. L; Rolh, E. F.Jr. Blood 1989, 74,1213-21. (2) Bunn, H. F.; Forget, B. G. Hemoglobin: Molecular, Genetic and Clinical Aspects; W. B. Saunders: Philadelphia, PA, 1986. (3) Shackleton, C.H.L.; Witkowska, H. E. In Mass Spectrometry: Clinical and Biomedical Applications; Desiderio, D. M, Ed.; Plenum: New York, 1994; Vol. 2, pp. 135-99. (4) Huisman, T.H.J. In The Hemoglobinopathies; Huisman, T.H J., Ed.; Churchill Livingstone, Edinburgh, 1986; pp. 1-31. (5) Rahbar, S. et al. Hemoglobin 1986, 10, 379-400. (6) Matsuo, T. et al. Biomed. Mass Spectrom. 1981,8,25-30. (7) Wada, Y.; Matsuo, T.; Sakurai, T. Mass Spectrom. Rev. 1989, 8, 379-434. (8) Lee, T. D.; Rahbar, S. In Mass Spectrometry of Peptides; Desiderio, D. M., Ed.; CRC: Boca Raton, FL, 1991; p. 257. (9) Katakuse, I. et al. Biomed. Mass Spectrom. 1984,11, 386-91. (10) Pucci, P.; Carestia, C; Fioretti, G.; Mastrobuoni, A. M.; Pagano, L. Biochem. Biophys. Res. Commun. 1985,130, 84-90. (11) Prome, D. et al. Biomed. Environ. Mass Spectrom. 1988,16,41-44. (12) Green, B. N. et al. In Biological Mass Spectrometry; Burlingame, A. L.; McCloskey, J. A., Eds.; Elsevier Science: Amsterdam, The Netherlands, 1990; pp. 129-46. (13) Ferrige, A. G.; Seddon, M. J.; Green, B. N.; Jarvis, S. A.; Skilling, J. Rapid Commun. Mass Spectrom. 1992, 6, 707-11. (14) Witkowska, H. E.; Bitsch, F.; Shackleton, C.H.L. Hemoglobin 1993, 77,227-42. (15) Covey, T. R.; Huang, E. C; Henion, J. D. Anal. Chem. 1991, 63, 1193-1200. (16) Juhasz, P.; Papayannopoulos, I. A.; Zeng, C; Papov, V.; Biemann, K. In Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics, May 31-June 5, 1992; Washington, DC, 1992; pp. 1913-14. (17) Rahbar, S.; Louis, J.; Lee, T.; Asmerom, Y Hemoglobin 1985, 9, 559-76. (18) Ferranti, P. et al. Biochem. Biophys. Acta 1993,1162,203-08. (19) Smith, R. D. et al. Biol. Mass Spectrom. 1993,22,112-20. (20) Bakhtiar, R.; Wu, Q.; Hofstadler, S. A.; Smith, R. D. Biol. Mass Spectrom. 1994, 23, 707-10. (21) Witkowska, H. E.; Green, B. N.; Morris, M.; Shackleton, C.H.L/ Mass Spectrom., in press. (22) Falick, A. M.; Witkowska, H. E.; Lubin, B. H; Nagel, R. L; Shackleton, C.H.L. In Techniques in Protein Chemistry II; Villafranca, J. J., Ed.; Academic Press: San Diego, CA, 1991; pp. 557-65. (23) Witkowska, H. E. et al. New Engl. J. Med. 1991,525,1150-54. Cedric H. L. Shackleton and H. Ewa Witkowska are members of the clinical MS group at Children's Hospital Oakland, which specializes in variant hemoglobin diagnosis. Address correspondence to Shackleton at Children 's Hospital Oakland Research Institute, 74752nd St., Oakland, CA 94609.
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