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Editorials |
Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch, New Zealand, and, Christchurch School of Medicine, University of Otago, E-mail steve.brennan{at}otago.ac.nz
Hemoglobinopathies are the archetypal molecular disease, and from 1949, when Linus Pauling et al. first demonstrated that hemoglobin (Hb) S could be separated from Hb A by paper electrophoresis (1), the detection and identification of Hb variants has provided a testing ground for new methods of protein analysis. The actual molecular basis of sickle cell anemia was not finally elucidated until 1959, when Vernon Ingram perfected the techniques of tryptic peptide fingerprinting (2). These modest beginnings led to the evolution of more sensitive analytical techniques, and the number of structurally characterized Hbs has now expanded to more than 1000 known variants.
During the 1960s and 1970s, 2-dimensional peptide mapping on paper and ion exchange mapping on Dowex resins led to the identification of many new variants until these techniques were eventually superseded by reversed-phase HPLC in the 1980s. Along with the introduction of reversed-phase chromatography, the need for more readily automated procedures for the quantification of HbA2 and HbF for thalassemia assessment and HbA1c in relation to diabetic control led to the introduction of cation exchange HPLC systems as a means of separating Hb species. Because thalassemia is the most common genetic disease world wide, and because of the prevalence of diabetes in western cultures, many ion exchange separations are performed annually, leading to the incidental detection of large numbers of "interfering" Hb variants. In the 1990s, refinements of soft-ionization techniques, such as fast atom bombardment, electrospray, and matrix-assisted laser desorption, heralded the introduction of protein mass spectrometry, and those working on Hb analysis were among the first to exploit the power of this new technology in a diagnostic setting.
In this issue of Clinical Chemistry, Kleinret and colleagues (3) provide an interesting account of their experience in the analysis of Hb samples from 2105 individuals referred to their laboratory over a 5-year period. Each sample was analyzed by cation exchange HPLC, reversed-phase HPLC, and mass spectrometry to detect variants with altered charge, hydrophobicity, or mass, respectively. The application of cation exchange chromatography showed quantitative variations in HbA2 and HbF, indicating some 204 cases of thalassemia, and the sequential use of ion exchange chromatography, reversed-phase chromatography, and mass spectrometry led to the detection of some 226 genetic variants of Hb. Of these, 4 contained substitutions (Asn
Ser, ThrSer, or GlySer) that lacked charge or hydrophobicity changes and could be detected only by mass spectrometry, and a further 4 contained variants detectable only by their hydrophobicity and mass differences. Although these statistics indicate that ion exchange alone would detect 218 of the 226 consecutive variants, it should be emphasized that the variants were not unique. An alternative interpretation of the data gives a better appraisal of the value of the combined approach. Of the 18 unique variants that were identified, the use of ion exchange alone detected 12, ion exchange plus reversed-phase detected 15, and the combined use of all 3 methods led to the detection of all 18 variants.
The traditional ion exchange and alkaline cellulose acetate electrophoresis methods are readily capable of detecting the majority of common variants, such as HbC, HbD, or HbE. However, the substitutions involved in these, and similar charge variants (Glu
Lys, GluGln, AspAsn, and LysGln) involve mass changes of 1 Da or less, and are not detectable by mass spectrometry. So the combined use of mass spectrometry with electrophoresis, or ion exchange, becomes very powerful because the procedures detect entirely different subsets of mutations. All the mutations reported by Kleinret et al. could have been detected by these 2 methods alone, so why include reversed-phase HPLC in the protocol? The practical answer to this question is revealed by Kleinret et al.’s comparative study of different types of mass spectrometer. Both electrospray ionization/quadrupole and MALDI-TOF instruments can resolve 50/50 mixtures of globin species if their mass difference is >6 Da. Therefore the allowable point substitution of AsnIle (–1 Da) would go undetected if only charge and mass were considered. Indeed, other mutations above the 6 Da threshold may also be missed if reversed-phase HPLC is not included in the analysis; those expressed in lower amounts, such as 
-chain variants, and some unstable, or thalassemic, β-chain variants. Another interesting observation was that Fourier transform ion cyclotron resonance mass spectrometry offered no practical advantage over the more modestly priced alternatives in relation to variant detection.
As well as providing insights from their practical experience of Hb analysis, Kleinret and colleagues asked the question, how many different variants are out there, and how many of them have been found? When they considered all possible point substitutions of both the - and β-globin genes, they found there were 1695 possible variants of adult Hb; 733 of which have been characterized to date. Considering each of the1695 theoretical substitutions individually, these investigators tabulated predications as to whether the variant would be detected by charge-, hydrophobic-, or mass-based assessments. These predictions are, however, of limited practical value because they fail to take into account: (a) posttranslational modifications such as the retention and partial acetylation of the –1 Met associated with the 1ValLeu substitution in Hb St Jozef (4), or possible deamidation consequent to the introduction of new Asn-Gly sequences, as occurs in Hb J Singapore (79AlaGly) and Hb J Sardegna (50HisAsn) (5); (b) conformational constraints that may suppress ionization, as occurs in Hb Volga (β27AlaAsp), preventing its detection by charge-based separations in the absence of denaturants (6); (c) changes in quaternary structure that effect the equilibrium constant between 1β2 dimers and tetramers and generate mixed hybrid bands on electrophoresis; and (d) heme loss and oxidation that make silent variants such as Hb Koln (B98ValMet) detectable by standard cellulose acetate electrophoresis (7).
The widespread application of HPLC for the detection of thalassemia and diabetes has been accompanied by a shift in the location of Hb analysis from hematology to biochemistry laboratories. We must not forget, however, the importance of a full blood count and an assessment of erythrocyte morphology in investigating a potential hemoglobinopathy. The results of these tests may suggest the presence of an accompanying thalassamia, which may explain an unusually high or low concentration of a variant. Simple heat- or isopropanol-stability tests are also of paramount importance when the Heinz body hemolytic anemias are under consideration. The majority of these unstable Hbs result from electrophoretically silent mutations within the hydrophobic core of the protein (6), and thus examination of the redissolved isopropanol precipitate by mass spectrometry can be very helpful in the search for a suspected unstable Hb (8). The pathological mutations that cause erythrocytosis are usually located at the subunit interfaces, within the heme pocket, or at the 2,3 diphosphoglycerate binding site. In these cases oxygen affinity or P50 measurements can point to, or more commonly exclude, the presence of a high-affinity variant.
Although the statistics indicate that some 43% of all possible and β variants have now been found, there are still plenty out there, because the predictions of Kleinret et al. considered only single amino acid replacements within the coding regions of adult Hb. One thing the study of Hb mutation has taught us is that anything that can go wrong will. When analyzing Hb we must also consider multiple amino acid insertions/deletions, frame shifts, stop codon mutations, and gene fusion products. Indeed even silent DNA mutations, such as CUG (Leu)CUC (Leu), are capable of changing the amino acid sequence by introducing exonic splice enhancers into the mRNA sequence and creating new exons (9). Surprisingly even DNA predictions can be wrong. In Hb Bristol the GTGATG DNA mutation predicts a β65ValMet substitution; however, the location of the new methoinine, above the distal histidine, leads to its immediate oxidation to aspartic acid in the mature protein (10). It is these exceptions that make the investigation of abnormal Hbs rewarding, from both a diagnostic and research point of view; and protein mass spectrometry provides the means of detecting these entities.
Interestingly mass spectrometry has also been successfully employed in a less familiar quantitative role, in screening for β thalassemia (11). The "bottom up" approach monitors secondary ions from 3 informative 
- and β-chain tryptic peptides and provides a 3-point surrogate assessment of the percentage of Hb A2 present. The high-throughput procedure has the advantage of being less prone to interference from coexisting genetic variants than the present HPLC or capillary electrophoresis methods and should provide a useful supplement to these methods in specialist reference centers for population screening.
Acknowledgments
Grant/funding Support: None declared.
Financial Disclosures: None declared.
References
Leu N-terminal mutation leading to retention of the methionine, and partial acetylation found in the globin gene in cis with a -3.7 thalassemia deletion. Hemoglobin 2007;31:313-323.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]50(CD8)HisAsnAsp]. Clin Chem 1999;45:2-28.Asp, a new highly unstable haemoglobin with a suppressed charge. FEBS Lett 1975;58:122-125.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]methionine): an unstable protein causing inclusion body anaemia. Nature 1966;210:915-916.[CrossRef][Medline]
[Order article via Infotrieve]37(C2)ProLeu]: a new mildly unstable mutation at an invariant proline residue. Hemoglobin 2002;26:389-392.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]Asp). Blood 1996;88:341-348.The following articles in journals at HighWire Press have cited this article:
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  R. H. Bateman, B. N. Green, and M. Morris Electrospray Ionization Mass Spectrometric Analysis of the Globin Chains in Hemoglobin Heterozygotes Can Detect the Variants HbC, D, and E Clin. Chem., July 1, 2008; 54(7): 1256 - 1257. [Full Text] [PDF]
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