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Technical Briefs |
(
1
Division of Clinical Chemistry, Department of Medical Laboratory Sciences and Technology and
3
Clinical Research Center, NOVUM, Huddinge University Hospital, SE-141 86 Stockholm, Sweden;
2
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden;
aauthor for correspondence: fax 46-8-585-812-10, e-mail britta.landin{at}chemlab.hs.sll.se)
Erythrocytosis, which is characterized by high hematocrit (packed cell volume), is a common condition causing complications attributable to hyperviscosity of blood. According to modern nomenclature, patients can be subdivided into one group of "apparent erythrocytosis" and one of "absolute erythrocytosis" (1). The absolute-erythrocytosis group contains both genetic disorders, acquired conditions such as polycythemia, and secondary reactions attributable to chronic hypoxemia or renal disease. The occurrence of hemoglobin (Hb) variants with increased oxygen affinity is a well-known cause of hereditary erythrocytosis, but in recent years interest has also been given to mutations in the erythropoietin receptor gene (2). Theoretically, functional studies of oxygen saturation curves would be the ideal method to screen for Hb variants that cause erythrocytosis, but such methods are not generally available and there are diverging opinions whether P50 measurements of stored or shipped samples can be used (3)(4). Of the nearly 200 Hb variants with increased oxygen affinity described, fewer than one-half have been found in conjunction with significant erythrocytosis (3). Several of the variants are attributable to amino acid substitutions that do not affect the net charge, i.e., the substitutions are "silent" when investigation is performed by conventional electrophoretic techniques. In contrast, almost all Hb variants in this group can readily be found by mass spectrometry exploiting the change in molecular weight of the variant globin.
Using electrospray mass spectrometry (ESMS), we have screened 70 consecutive samples from patients with unexplained erythrocytosis that had showed normal Hb pattern on isoelectric focusing (IEF) (5) and HPLC (6)(7) analysis. The investigation was approved by the Ethics Committee at Huddinge University Hospital. In three cases, we detected ß-globin variants. This report illustrates how the information from electrophoretic methods and the knowledge of the wild-type globin gene sequence can facilitate the interpretation of mass spectral findings.
The database of human Hb variants found at the Globin Gene Server home page (8) was searched for Hb variants associated with erythrocytosis. By this means, 89 different Hb variants associated with erythrocytosis were found. [Details of the results of the database search are available as a data supplement at Clinical Chemistry Online (http://www.clinchem.org/content/vol47/issue7/)]. Of these variants, 33 were neutral, i.e., the substitution involved amino acids of the same group (neutral, basic, or acidic). In 9 of the 89 variants, the mass of the variant did not differ from that of the wild-type globin by >6 Da. Of this group of variants, which as the intact protein would not be expected to be resolved from wild-type globin by ESMS on quadrupole instruments, only one neutral Hb variant (Hb Linköoping, ß36 Pro
Thr) was found. Although this variant cannot be separated from Hb A0 by conventional acid electrophoresis, IEF has shown excellent separation (9).
ESMS analysis was performed on two different instruments capable of resolving intact Hb masses that differ by >6 Da with the aid of a deconvolution program. The initial screening of the series of whole-blood samples was carried out using a Quattro I mass spectrometer (Micromass), and the trypsin-digested (Sigma-Aldrich) samples were analyzed on a quadrupole time-of-flight mass spectrometer (Micromass) as described previously (10). A 1612-bp DNA fragment including all three exons of the ß-globin gene was amplified by PCR using primers 5'-ATAAGTCAGGGCAGAGCCATCTAT-3' and 5'-TTAGGCAGAATCCAGATGCTCAAG-3'. Nucleotide sequencing of exons 1 and 2 was performed using a sequencing primer (5'-GGAATTCCCATTTGCTTCTGACACAACT-3') and Big DyeTM terminator (PE Applied Biosystems).
The mass spectra of intact globins displayed peaks for the wild-type 
(15 126 Da) and ß (15 867 Da) chains and their common adducts (Na+, K+, heme, and glutathione). ESMS analysis of the first case showed the presence of a ß variant (ßX) with a mass shift of +32 Da from the wild-type ß chain (Fig. 1A
). The variant constituted 
54%. Several possible amino acid substitutions correspond to a +32 Da mass difference: AlaCys, AspPhe, MetTyr, ProGlu, and ValMet. Only the ValMet substitution, however, results from a single-base substitution. This is also a neutral substitution. In the ES mass spectrum of the tryptic digests, an intense doubly protonated peak of wild-type ßT3 (m/z 657.8) was observed as well as a variant peak (m/z 673.8) 16 Thompson above (Fig. 1A
, inset). This corresponds to a +32 Da shift in the uncharged molecule. The location of a variant peptide ßT3 was further supported by observation of a peak corresponding to the singly protonated variant peptide (m/z 1346.7) 32 Thompson above the wild-type ßT3 peptide peak (m/z 1314.7).
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To pinpoint which of the three valine residues in the ßT3 peptide (VNVDEVGGEALGR) was substituted, the doubly protonated wild-type and variant peptides were subjected to tandem MS (MS/MS). The MS/MS spectra for both peptides showed almost complete series of singly charged Y'' ions (Y''2–Y''12) as well as complementary B ions (B2–B7; Fig. 1B
). The observation of the same m/z value for the Y''8 ion in the MS/MS spectra of both the wild-type and variant peptides rules out substitution of the valine, which is the eighth residue from the COOH terminus. The mutation was localized by the Y''11 and B3 fragment ions, which were shifted by +32 Thompson from the corresponding Y''11 and B3 ions of the wild-type peptide. The effect of this m/z change attributable to the ValMet substitution on the consecutive fragments was demonstrated by an equivalent m/z shift in Y''12 and were supplemented by an equal m/z shift in B3 to B6 ions. The ValMet substitution located by Y''11 and B3 corresponds to position 20 in the ß-globin chain, indicating the substitution ß20ValMet. Thus, the variant is identified as Hb Olympia (ß20ValMet) (11). Sequence analysis of amplified DNA confirmed the expected mutation GA in codon ß20. In a similar way, a second case of Hb Olympia was identified in this investigation.
The ES mass spectrum of the intact globin chains from the third case showed a ß variant (ßX) 14 Da larger than the wild-type ß-globin (Fig. 1C
). The variant constituted 51%. There are eight possible amino acid exchanges that correspond to the mass shift of +14 Da: AsnGln/Lys, AspGlu, GlyAla, SerThr, ThrAsp, and ValLeu/Ile. The AsnGln and AsnLys substitutions are not possible as single point mutations and thus were ruled out. Of the remaining possibilities, a ThrAsp substitution would be expected to alter the intrinsic charge of the Hb molecule, which was not compatible with the IEF pattern. Considering the remaining five possible substitutions and starting with the already described Hb variants associated with erythrocytosis (see on-line data supplement), we directed our interest to tryptic peptides ßT11 and ßT12, both of which represent inner core peptides of the Hb molecule. Because the core peptides often are difficult to characterize by ESMS (12), DNA sequencing was performed. Heterozygosity for a mutation TA was found, corresponding to ß99 Asp (GAT)Glu (GAA). We then performed ESMS to confirm this mutation on the protein level, our interest being focused on the ßT11 peptide (LHVDPENFR). Analysis of the tryptic digest demonstrated doubly protonated wild-type (m/z 563.8) and variant (m/z 570.8) peptides as shown in the inset of Fig. 1C
. The 7-Thompson change in m/z in the doubly protonated peptide is equivalent to a change of 14 Da in the intact ß chain.
MS/MS analysis of the doubly protonated wild-type and the variant peptides localized the amino substitution to the Y''6 and B4 ions corresponding to residue 99 in the wild-type ß chain (Fig. 1D
). Both the ESMS and DNA sequencing conclusively indicated the substitution ß99AspGlu, which is known as Hb Coimbra (13) or Hb Ingelheim (14).
In this study, three cases of Hb variants known to cause erythrocytosis were found by ESMS but missed by the IEF method. In two cases, the diagnosis of heterozygous Hb Olympia (ß20 ValMet) was achieved solely by the use of ESMS. This could be accomplished because the array of possible substitutions was limited by the assumptions that the neutral variant had been described previously and was attributable to a point mutation. In the third case, the initial mass spectral findings indicated a variant ß-chain and provided preliminary data. Because these were compatible with several substitutions in the inner core of the Hb molecule, DNA sequencing was performed and revealed heterozygosity for Hb Coimbra (ß99 GATGAA). The DNA result was later confirmed on the protein level by ESMS and MS/MS experiments.
Although Hb variants are relatively seldom found to be the cause of high Hb concentrations in unselected patient groups, the possible occurrence of a Hb variant often is considered early in the investigation of patients with high Hb concentrations. Standard techniques commonly used for screening for Hb variants, such as electrophoresis, IEF, or ion-exchange HPLC, are not sufficient because 33 of the 89 different Hb variants associated with erythrocytosis do not induce any major change in net charge of the Hb molecule. The additional use of ESMS is encouraged because combination of this technique and IEF will increase the sensitivity for Hb variants that cause erythrocytosis to 100%.
Acknowledgments
This study was supported by the Swedish Medical Research Council (Grant 03X-12551) and the research funds of Karolinska Institutet. We thank Gertrud Pfuhl Bertilsson for performing the DNA sequencing.
References
Thr): a new hemoglobin mutant characterized by reversed-phase high-performance liquid chromatography.. J Chromatogr 1984;297:31-36..[ISI][Medline]
[Order article via Infotrieve]
-chain haemoglobin variants. Rapid Commun Mass Spectrom 2000;14:1184-1194..[ISI][Medline]
[Order article via Infotrieve]
methionine): an electrophoretically silent variant associated with high oxygen affinity and erythrocytosis.. J Clin Invest 1973;52:342-349..
2ß299(G1)AspGlu, a newly discovered high oxygen affinity variant. Hemoglobin 1991;15:487-496..[ISI][Medline]
[Order article via Infotrieve]
Glu]: a new high affinity hemoglobin variant destabilizing the 1ß2 interface [Abstract]. Blood 1991;78(Suppl 1):206a.The following articles in journals at HighWire Press have cited this article:
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  F. Boemer, O. Ketelslegers, J.-M. Minon, V. Bours, and R. Schoos Newborn Screening for Sickle Cell Disease Using Tandem Mass Spectrometry Clin. Chem., December 1, 2008; 54(12): 2036 - 2041. [Abstract] [Full Text] [PDF]
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R. W. Burnett Screening for Hemoglobin Variants with Abnormal Oxygen Affinity Clin. Chem., February 1, 2002; 48(2): 391 - 391. [Full Text] [PDF]
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