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Clinical Chemistry 54: 69-76, 2008. First published October 11, 2007; 10.1373/clinchem.2007.089961
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(Clinical Chemistry. 2008;54:69-76.)
© 2008 American Association for Clinical Chemistry, Inc.

Hematology

Mass Spectrometry: A Tool for Enhanced Detection of Hemoglobin Variants

Peter Kleinert1, Marlis Schmid2, Karin Zurbriggen2, Oliver Speer2, Markus Schmugge2, Bernd Roschitzki3, Silke S. Durka1, Urs Leopold1, Thomas Kuster1, Claus W. Heizmann1, Hannes Frischknecht4 and Heinz Troxler1,a

Divisions of1 Clinical Chemistry and Biochemistry and 2 Hematology, Department of Pediatrics, University of Zurich, Zurich, Switzerland; 3 Functional Genomics Center Zurich, Zurich, Switzerland; and 4 IMD Institute for Medical and Molecular Diagnostics Ltd., Zurich, Switzerland.

aAddress correspondence to this author at: Department of Pediatrics, University of Zurich, Division of Clinical Chemistry and Biochemistry, Steinwiesstrasse 75, 8032 Zurich, Switzerland. Fax 41-44-266-71-69; e-mail heinz.troxler{at}kispi.unizh.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: More than 900 hemoglobin (Hb) variants are currently known. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. In our laboratory, we routinely apply chromatographic methods. To ascertain whether Hb variants are missed with our procedures, we additionally analyzed all samples with mass spectrometry (MS).

Methods: Database evaluation was performed using all entries made in the Hb variant database HbVar, and possible Hb variants were calculated based on DNA variations. During a 5-year period, we analyzed 2105 lysates with cation-exchange HPLC (PolyCAT A column) and reversed-phase HPLC and additionally with electrospray ionization or MALDI-TOF MS. Globin chains were identified by their molecular masses.

Results: Database evaluation revealed that 43.2% of all possible Hb{alpha}- and β-chain variants were found to date (considering only single-point mutations). Currently, 68.2% of the possible charge difference variants and only 28.7% of the neutral variants are found. Among 2105 Hb samples we identified 4 samples with Hb variants that were detected only with the MS method; 2 were new Hb variants (Hb Zurich-Hottingen and Hb Zurich-Langstrasse). With cation-exchange HPLC, 1 sample was found to be a β-thalassemia and was identified by MS to be a β-variant (Hb Malay). More common variants, such as Hb C, Hb D, and Hb E, and thalassemias could not be detected with the MS method.

Conclusions: Application of MS improves the sensitivity of Hb analysis. The combination of MS with electrophoretic and chromatographic methods is optimal for the detection of Hb variants.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hemoglobinopathies are a group of disorders affecting red blood cells with abnormal hemoglobin (Hb).1 Thalassemias, in contrast, are characterized by impaired synthesis of normal globins (1)(2). Structural Hb variants are typically due to a point mutation in a globin gene that produces a single amino acid substitution in a globin chain. Although most of these variants are of limited clinical significance, many of them, such as homozygous Hb S and Hb C, produce severe clinical manifestations. Furthermore, variants with no clinical manifestations could cause substantial overestimation of HbA1c, the important marker for diabetes mellitus. Worldwide, an estimated 150 million people carry Hb variants (3), and hemoglobinopathies are the commonest inherited disorders, constituting a significant healthcare problem (4). Therefore, reliable detection and identification methods are essential.

Since the pioneering study of Linus Pauling in 1949 (5), in which the different electrophoretic mobilities of sickle cell anemia Hb and normal Hb were described, more than 900 Hb variants have been identified and listed in the globin gene server (HbVar) database (6)(7). In this database, Hb variants leading to a charge difference are significantly overrepresented compared with neutral Hb variants. This result is surprising, because only 5 of the 20 amino acids contain either a basic (Lys, Arg, His) or an acidic (Asp, Glu) side chain, whereas the other 15 amino acid side chains are uncharged. Thirty-six of 141 amino acids in the {alpha}-chain and 38 of 146 residues in the β-chain are charged residues. This disproportion is explained by the characteristics of the analysis technique used; electrophoresis at alkaline or acidic pH has been the methods of choice for Hb analysis, although chromatographic assays were also widely used (8)(9)(10)(11).

Reversed-phase (RP) and cation-exchange HPLC are often applied, and the latter enables the quantification of various Hb fractions, including HbF and HbA2, along with Hb variants (2)(10). Therefore, this method is well suited for the detection of hemoglobinopathies and some thalassemias. The separation power of the electrophoretic methods is mainly dependent on the charge state of the Hb variant, and in cation-exchange HPLC, separation is due to the affinity of the Hb cations to the polyaspartic acid-coated stationary phase. In contrast, in RP HPLC, the separation is based on the hydrophobicity of the Hb chains.

During the last 3 decades, mass spectrometry (MS), a technique that is widely used in clinical chemistry, laboratory medicine, and research, found its way into the field of Hb analysis. In 1981, Wada et al. (12) pioneered the analysis of tryptic peptides of Hb by MS. The development of the soft ionization techniques electrospray ionization (ESI) and MALDI made it possible to use MS to study intact globin chains. In 1990, the 1st application of ESI MS involving intact Hb chains was reported by Falick et al. (13). ESI or MALDI-TOF MS is now becoming a common auxiliary method applied in routine Hb analysis. The use of MS techniques has led to the discovery of more than 60 new mutations (14), and even the intact Hb tetramer can be analyzed using a nanoESI MS technique (15). Furthermore, MALDI-TOF MS is a highly sensitive method that enables the analysis of Hb chains from a single red blood cell (16). Final identification of a variant is achieved either by molecular biology techniques or by protein sequence analysis, in which MS now also occupies a key position (17). In(18), variants with mutation sites close to the termini of the β-chain were identified by ESI MS/MS of the intact Hb chain.

In this study, we calculated the pI, the hydrophobicity index, and the mass difference of detected (entries in the HbVar database) and undiscovered (theoretically possible) Hb {alpha}- and β-variants. We statistically assessed the incidence of charge-, hydrophobicity-, and mass-difference variants. Experimentally, we used MS to investigate whether Hb variants are missed when 2 chromatographic techniques (cation-exchange and RP HPLC) are applied for Hb analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
database analysis and calculations
All entries made in the globin gene server (HbVar) database (7) until July 19, 2007, were included in this study, but only exonic single-point mutations leading to a single amino acid change are considered. Also, effects of protein processing aberrations in Hb variants, e.g., in Hb Marseille or Hb Raleigh, were disregarded. The pIs of the protein variants were calculated using the method of Bjellqvist et al. (19) with the software accessible on the ExPASy homepage (http://www.expasy.ch). Variants with a pI difference >0.050 units compared with the pI of the normal chains were considered as charge difference variants. Hydrophobicity values were calculated using the method described by Kyte and Doolittle (20). Variants with hydrophobicity values that differ more than 0.010 units from the normal chains were considered as hydrophobicity change variants.

blood samples
During a 5-year period, 2105 blood samples from different patients were collected with EDTA or heparin as anticoagulant, stored at 4 °C, and analyzed within 4 h. Hemolysates were prepared for chromatographic analysis by lysing washed erythrocytes in 1 mmol/L KCN.

hB analysis with hplc and ief
We performed cation-exchange HPLC (PolyCAT A column) according to (21) and RP HPLC as described in (22). Samples that were found with chromatographical methods to be abnormal were confirmed by IEF according to (23).

esi and maldi-tof ms
A PerkinElmer SCIEX API 365 LC/MS/MS system equipped with a PerkinElmer Series 200 autosampler was used for ESI MS analysis. Mass spectra were acquired in the range of m/z 700 to 1800, with step size 0.1. Samples were diluted 1:150 in acetonitrile-water (50:50 by volume) containing 1 mL CH3COOH/L; 10 µL of the diluted lysate samples was injected. An autoflex® system (Bruker Daltonics®) was used for MALDI-TOF MS of the lysate samples. The mass resolution at 15 kDa was approximately 750 (m/{Delta}m, FWHM). Samples were diluted 1:150 in acetonitrile-water (50:50 by volume) containing 1 mL CH3COOH/L. The samples were analyzed by the overlayer method. A thin layer of sinapinic acid solution (saturated solution in ethanol) was applied to a ground-steel MALDI target. Equal volumes of protein sample and a saturated sinapinic acid solution (containing, per liter, 330 mL CH3CN and 1 mL trifluoroacetic acid) were mixed, and 0.5 µL of the mixture was applied to the thin layer and dried at room temperature.

We analyzed 57% of the samples with ESI MS and 43% of the samples with MALDI-TOF MS.

dna analysis
DNA was prepared from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen) and amplified using 2 specific primer sets for the β-globin gene (sense primer, 5'-GCAATTTGTACTGATGGTATGG-3', and antisense primer, 5'-CCACACTGATGCAATCATTCG-3'). PCR amplification was performed using a DNA thermal cycler (Catalys AG) in a 50-µL reaction volume containing 100 ng genomic DNA, 1x polymerase mix (Eppendorf MasterMix 2.5x, Vaudaux-Eppendorf), and 0.5 µmol/L of primers. Amplification was performed with an initial heat activation step of 2 min at 95 °C followed by 35 cycles of 95 °C for 45 s, 54 °C for 45 s, and 72 °C for 90 s and then an extension step at 72 °C for 10 min. The amplified DNA product was sequenced using the same primers with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems).

high-resolution ms
Ion Trap and Fourier Transform Ion Cyclotron Resonance MS was performed on an LTQ-FT mass spectrometer (Thermo Electron) using the NanoMateTM 100 with ESI Chip Technology (Advion Biosciences). The AGC target values were set to 7 x 105 for full scan mode. A resolving power of 200 000 at 400 m/z was used in acquisition of MS spectra, and 1 scan consisted of 40 microscans. Hb samples were diluted 1:150 in methanol-water (50:50 by volume) containing 0.2% formic acid. A sample volume of 15 µL was delivered to the MS using the NanoMateTM 100 system. A NanoMate high-density ESI chip was used as static nanoelectrospray emitter, providing stable spray conditions. Voltages of 1.3–1.6 kV were applied to the chip through the NanoMate power supply, whereas the mass spectrometer source voltage was set to 0. Samples were infused using nitrogen gas at a pressure of 4000 Pa (0.6 psi).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
database evaluation and calculations
To obtain the proportion of detected vs not yet detected variants, we calculated, on the basis of the DNA, every possible protein variant (only single-point mutations in the exons leading to a single amino acid substitution) of the {alpha}- and β-globin chains. The results are summarized in Fig. 1 and in Tables 1 ({alpha}-chain) and 2 (β-chain) in the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol53/issue12 . The {alpha}-chain with 141 amino acids gives rise to a total of 1269 DNA variants. Because of the degenerated DNA code, 834 different protein variants are possible. To date (cutoff date: July 19, 2007), 295 {alpha}-variants (35.4%) are known. The evaluation of the β-chain revealed 861 possible protein variants (1314 DNA variants), of which 438 have been observed (50.9%). Taking the {alpha}- and β-variants together, 43.2% of the possible variants have been detected and 962 variants (56.8%) have not (Fig. 1A ).


Figure 1
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  		Figure 1. Evaluation of all possible and detected protein variants (cutoff date: July 19, 2007; only single point mutations are regarded).

A total of 834 variants of the {alpha}-chain, 861 variants of the β-chain, and 1695 of both chains are possible. A shows the numbers of all possible variants, whereas in B and C, the number of variants differentiated according to their calculated charge are shown. Variants with pI differences of ≥±0.050 units were considered as charge-difference variants.

To assess whether an Hb variant should be detectable with electrophoretic or chromatographic methods, we calculated the pI (19) and the hydrophobicity indices (20) of all Hb {alpha}- and β-variants (see Tables 1 and 2 in the online Data Supplement). pI differences of ≥±0.050 units were considered as charge-difference variants. Calculations revealed 290 possible {alpha}-variants with a charge difference. Currently, 173 (59.7%) have been detected (Fig. 1BUp ). On the other hand, only 122 of 544 neutral variants (22.4%) are known (Fig. 1CUp ). In the β-chain, 253 of 335 (75.5%) possible charge difference variants have been found, and only 185 of the 526 possible neutral variants (35.2%) have been detected. Overall, in 763 of 962 undetected variants we calculated a pI-shift of <0.050 units. For these variants we would expect no differences in electrophoretic mobilities.

To evaluate whether the calculated pI change is a useful predictive parameter for the detectability in electrophoretic or chromatographic methods, we analyzed the entries of the known {alpha}- and β-variants in the HbVar database and compared the information given in the "Electrophoresis" and "Chromatography" sections with the calculated pIs. In cellulose acetate electrophoresis and anion-exchange HPLC, the calculated pI change predicted correctly a mobility difference in approximately 85% and 90% of all variants, respectively, and in cation-exchange HPLC and IEF, the calculated pI difference gave a correct prediction in 70% and 63% of the variants, respectively.

Considering these factors, we would expect that approximately 670 and 710 of the 962 not-yet-detected variants are silent in cellulose acetate electrophoresis and anion-exchange HPLC, respectively. Approximately 590 and 550 variants would be expected to be silent in cation-exchange HPLC and IEF.

We also calculated the hydrophobicity values for each variant, and a difference of ±0.010 units was considered as a hydrophobicity change; 41.2% of the hydrophobicity change variants and 45.0% of the nonchange {alpha}- and β-variants have been detected. Evaluation of the HbVar database entries revealed that the hydrophobicity difference correctly predicts the detectability in RP HPLC in 62% of the variants. We therefore concluded that the calculated hydrophobicity values are inadequately predictive parameters.

Furthermore, calculations of the mass differences of all variants revealed that from the 962 currently undetected {alpha}- and β-variants, 92.0% (885 variants) would be detectable with MS. Mass differences of the globin chains of ≥6 Da are detectable with conventional mass spectrometers.

Evaluation of the number of substitutions of the {alpha}- and β-chain revealed that some of the most frequently possible variants were rarely found, such as in the {alpha}-chain Leu->Gln (15 possible variants, 0 detected) and Ala->Ser (21, 2), or in the β-chain Ala->Ser (15, 1) and Ala->Gly (15, 2). On the other hand, some substitutions are frequently found, such as His->Arg (10, 8) or Lys->Asn (11, 11) in the {alpha}-chain or Ala->Asp (14, 13) and Gly->Asp (12, 12) in the β-chain. A complete list of all substitutions is given in Table 3 in the online Data Supplement.

lysate analyses
Among the 2105 samples analyzed with cation-exchange HPLC (PolyCAT A column), RP HPLC, and ESI or MALDI-TOF MS in our pediatric test center during 5 years, 443 samples were found to be abnormal owing to variant Hbs, thalassemias, or increased minor fractions (samples with increased HbF excluded; Table 1 ). We found 4 chromatographically silent samples with the variant Hb Riccarton (2 unrelated patients) and 2 new variants: Hb Zurich-Hottingen and Hb Zurich-Langstrasse (1 patient each). One patient with Hb Malay was identified with cation-exchange HPLC to have β-thalassemia, and MS revealed the mutation in the β-chain. Two variants, Hb Hamilton and Hb City of Hope (2 patients), were silent in PolyCAT A HPLC but detected with RP HPLC and MS. On the other hand, the following variants were not detectable with MS: Hb C, D, E, E-Saskatoon, O-Arab, Lepore–Boston–Washington, and Schlierbach. Furthermore, the thalassemias and Hb H/Barts are not detectable with MS. Increased concentrations of HbA1c were detectable only when this variant consisted of more than 10%–15% of total Hb.


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  		Table 1. Results of lysate analyses.

In MALDI-TOF MS, the experimental precision of the mass measurement of the β-chain [M+H]+ was determined to be 0.005% (CV; n = 61; mean 15 869.110 Da; range 15 867.991–15 870.274 Da). The ESI MS precision was comparable to the MALDI-precision.

identification of hB zurich–hottingen and hB zurich-langstrasse
During this study, 2 new chromatographically silent Hb variants, Hb Zurich-Hottingen (24) and Hb Zurich-Langstrasse, were detected. Both showed no abnormalities as analyzed by cation-exchange HPLC, RP HPLC, or IEF. Hb Zurich-Langstrasse was detected in an 83-year-old woman who was hematologically asymptomatic. Her erythrocyte indices were as follows: Hb, 118 g/L; erythrocytes, 4.0 x 1012/L; hematocrit, 0.37 L/L; mean cell volume, 89 fL; mean cell Hb, 30 pg. HbA2 was 2.5% and HbF was 0.7%. The P50 (O2) was not determined. Fig. 2 shows the MALDI-TOF mass spectrum of the globins in Hb Zurich-Langstrasse. It exhibits a broad signal containing 2 barely resolved peaks corresponding to the normal β- and the mutated βx-chain with a mass difference of 14 Da. In the inset (Fig. 2 ), the DNA analysis of the β-gene is presented. It shows a heterozygous ACT->TCT mutation at codon 50, resulting in a Thr->Ser substitution in the β-chain.


Figure 2
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  		Figure 2. MALDI-TOF MS of Hb Zurich-Langstrasse.

The spectrum shows 3 intense peaks corresponding to the wild-type {alpha}-chain (15 126 Da) and β-chain (15 868 Da) and the variant βx-chain (15 854 Da). The mass difference of 14 Da is due to the Thr->Ser substitution in the β-chain. Peaks marked with an asterisk correspond to matrix adducts. The inset shows the DNA analysis of the β-gene using the forward primer. The mutation site (ACT->TCT at codon 50) is marked with an arrow.

high-resolution ms
To elucidate whether high-resolution MS enables detection of variants with low mass differences (<2 Da), we analyzed a control sample, a heterozygous Hb D sample, an Hb Lepore–Boston–Washington sample, and an Hb Malmö sample. The high-resolution MS of m/z = 934 (corresponding to the 17-fold protonated β-chain) are shown in Fig. 3 . The resolution was 200 000 at 400 m/z, indicating that differences of 1 Da are completely separated, and the different signals correspond to the isotopic pattern of the β-chain. Fig. 3A shows the MS of the control sample with a normal β-chain, and Fig. 3B shows the MS of the Hb A/D sample, where the molecular mass of the βD-chain is 1 Da less owing to the 121Glu->Gln substitution. Fig. 3C and 3D exhibit the signals of the β-chain in heterozygous Hb Lepore–Boston–Washington (βLepore-chain has a molecular mass shift of –2 Da compared with the normal β-chain) and of a patient heterozygous for Hb Malmö (mass shift –9 Da due to the 97His->Gln substitution), respectively. The mass spectra in Fig. 3 , A–C, are not distinguishable, whereas in Fig. 3D 2 β-chains are clearly detectable.


Figure 3
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  		Figure 3. High-resolution mass spectra of the 17-fold protonated β-chain ([β+17H+]17+) of a control lysate sample (A), a heterozygous Hb A/D sample (B), a heterozygous Hb Lepore–Boston–Washington sample (C), and a heterozygous Hb Malmö sample (D).

The resolution was 200 000 at 400 m/z; therefore, a completely resolved isotopic pattern of the signals was obtained.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was initiated by the observation that the predominant Hb variants listed in the globin gene server (HbVar) database are charge-difference variants, a feature attributable to historical and methodological reasons and clearly demonstrated by the evaluation of the number of amino acid substitutions in the {alpha}- and β-chain (see Table 3 in the online Data Supplement). The majority of the frequently detected substitutions are charge-difference substitutions. Calculation of the pIs (see Tables 1 and 2 in the online Data Supplement) revealed that two-thirds of the possible charge-difference variants are detected, whereas <30% of the neutral variants were found to date (Fig. 1Up ). However, a calculated pI-shift does not necessarily result in a different electrophoretic or chromatographic mobility of the variant. It must be considered that the 3-dimensional structure of the globins is determined principally by the residues that form the interhelical and helix-heme packings (25), and substitutions in these sites may lead to conformational changes in the proteins. The substitution effect also depends on the 3-dimensional position, viz. internal or external. For example, the {alpha}-variant Hb Sun Prairie (130Ala->Pro) is silent in IEF, whereas Hb Fontainebleu (21Ala->Pro) is detectable. The substituted amino acid is internal in Hb Sun Prairie and external in Hb Fontainebleu (7). As a very simple model, the calculation of the pI-shifts does not consider conformational changes that might alter the mobility. Therefore, mutations leading to a distinct conformational change can diverge from the predicted behavior. Furthermore, the model cannot predict reliably unstable variants.

Nevertheless, pI calculations and the evaluation of the method-specific detectability allow the prediction of the number of the currently undetected, silent variants. We estimate that from the undetected 962 variants (Fig. 1AUp ) a large proportion is silent with the most common methods, e.g., approximately 590 variants with cation-exchange HPLC or 670 variants with cellulose-acetate electrophoresis. Although this is only a rough estimate and may diverge in practice, we conclude that other methods that are not based on electrophoretic or chromatographic mobility should be applied in Hb variant analysis. Therefore, we suggest MS as a supplementary method. ESI and MALDI-TOF MS are methods that enable the detection of variants when the mass difference between the abnormal and the wild-type globin chains exceeds ±6 Da. As calculated in this study, MS methods would be able to detect 92% of the undetected variants (885 of 962 variants).

Experimentally, we investigated whether we miss neutral variants when using only chromatographic methods (cation-exchange and RP HPLC). Indeed, with additional MS analysis of all lysate samples during a period of 5 years, we detected 2 new variants, Hb Zurich-Hottingen and Hb Zurich-Langstrasse (Fig. 2Up ). Neither variant had a clinical impact. Furthermore, 2 samples with Hb Riccarton were detected. These neutral variants are exclusively found by MS and are chromatographically silent (see Table 1Up ). In an Hb Malay sample, only the MS analysis revealed the variant β-chain, whereas in cation-exchange HPLC this sample was identified as a β-thalassemia. Recapitulating, we would have missed 4 of 2105 samples (0.2%) or 1% of the abnormal samples without the use of MS analysis.

In this study, we applied the 2 most important MS techniques for protein analysis, ESI MS and MALDI-TOF MS. Our conventional instruments would allow the detection of variants with mass differences >6 Da and showed comparable mass resolution. In ESI MS, the sample preparation is very simple and requires only the dilution of the lysate sample. In contrast, in the MALDI-TOF MS approach, the lysate sample must also be diluted and then prepared according to the overlayer method as described in Materials and Methods, but the mass analysis of the globin chains is very fast and requires only a few seconds per sample.

Two important drawbacks of the MS methods should be mentioned. First, its insufficient resolution prevents the detection of Hb mutations with small mass differences of the globin chains. The precision of normal low-resolution mass measurements was insufficient to distinguish the wild-type β-chain from several β-chain variants, such as Hb C, D, or E. As shown in Fig. 3Up , owing to the isotopic pattern, even high-resolution MS did not separate globin chains that differed only in 1 or 2 Da from the normal chains. This result confirms the statement of Rai et al. (26) that high resolution per se does not appear to offer a solution. Furthermore, this result indicates that modestly priced instruments are just as useful in Hb analysis. Two intact globin chains are not observed as separate entities in MS unless their masses differ from one another by more than 6 Da (27).

Second, MS as described here is only a qualitative technique, and in particular, minor Hb fractions such as HbA1C or HbA2, which are important for diagnosis of diabetes mellitus or thalassemias, respectively, cannot be quantified.

Nevertheless, MS allows the detection of electrophoretically or chromatographically silent Hb variants and complements traditional techniques. Therefore, we consider MS an indispensable tool that contributes significantly to improved sensitivity in Hb analysis.


   Acknowledgments
 
Grant/funding Support: None declared.

Financial Disclosures: None declared.

Acknowledgments: We are grateful to Dr. Rowena Crockett for critical reading of the manuscript.


   Footnotes
 
1 Nonstandard abbreviations: Hb, hemoglobin; RP, reversed-phase; MS, mass spectrometry; ESI, electrospray ionization.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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