Phenotype determination of hemoglobinopathies by mass spectrometry

Available online at www.sciencedirect.com

Clinical Biochemistry 42 (2009) 1807 – 1817

Review

Phenotype determination of hemoglobinopathies by mass spectrometry Isabelle Zanella-Cleon a , Philippe Joly b , Michel Becchi a , Alain Francina b,⁎ a

b

Institut de Biologie et de Biochimie des Protéines, CNRS UMR 5086, IFR128, Université de Lyon, Lyon, France Unité de Pathologie Moléculaire du Globule Rouge, Fédération de Biochimie et de Biologie Spécialisée, Hôpital Edouard Herriot, Hospices Civils and Université Claude Bernard-Lyon 1, Lyon, France Received 15 April 2009; accepted 20 April 2009 Available online 3 May 2009

Abstract Objectives: Nowadays, nearly 1000 hemoglobin (Hb) variants are known. The standard biochemical techniques used in Hb analysis are mainly: isoelectric focusing, cation-exchange liquid chromatography (LC) and reversed-phase LC. In addition to this approach, a protein analysis is achieved by mass spectrometry (MS) and additional DNA studies are performed. The aim of this review is to emphasize the significance of MS methods applied to Hb variants analysis. Results and perspectives: To perform Hb studies, different MS techniques are currently used such as electrospray ionization (ESI), matrixassisted laser desorption ionization (MALDI) and tandem mass spectrometry (MS/MS). As shown here, MS is an efficient tool for identification of all types of variants (substitution of a single amino acid residue, several substitutions in the same globin chain, insertions/deletions, fusion Hbs). The use of MS in neonatal screening of Hb variants is also presented. Conclusions: MS is a powerful technique for Hb analysis. It appears as being an important additional method in the set of biochemical techniques. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Hemoglobin variant; Mass spectrometry; Electrospray ionization mass spectrometry; Matrix-assisted laser desorption mass spectrometry; Tandem mass spectrometry

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS methods in Hb variant analysis . . . . . . . . . . . . . . . . . . . . . . . Molecular weight determination of globin chains. . . . . . . . . . . . . . . Sample preparation for ESI-MS and MALDI-MS analysis . . . . . . . . MS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS analysis of globin chain-derived peptides . . . . . . . . . . . . . . . . Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS analysis of globin chain-derived peptides . . . . . . . . . . . . . . Screening methods using MS/MS of globin tryptic peptides . . . . . . . . . Problems in MS: cysteinyl residues. . . . . . . . . . . . . . . . . . . . . . Hemoglobin variants with a single mutation in the globin chain . . . . . . . . . Hemoglobin variants with two amino acid substitutions in a single globin chain Hemoglobin variants with an insertion or a deletion in a globin chain . . . . . . Hemoglobin variants with post-translational modifications . . . . . . . . . . . . Unstable hemoglobin variants . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. Unité de Pathologie Moléculaire du Globule Rouge, Fédération de Biochimie et de Biologie Spécialisée, Hôpital Edouard Herriot, 5, Place d'Arsonval, 69437 Lyon Cedex 03, France. Fax: +33 4 72 11 05 98. E-mail address: [email protected] (A. Francina). 0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.04.010

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Fusion hemoglobin variants . . . . . . . . . . . . . . . . . Hemoglobin variants with low expression . . . . . . . . . . Hemoglobin variants giving high molecular weight species . Embryonic and fetal hemoglobins . . . . . . . . . . . . . . MS and neonatal diagnosis of hemoglobinopathies . . . . . Minor components: Hb A2 and delta-chain variants. . . . . Methemoglobins . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction For analytical or preparative purposes, hemoglobin (Hb) variants are commonly separated from normal hemoglobins by electrophoretic and ion-exchange chromatographic techniques [1,2]. However, the efficiency of these techniques depends on the presence of a charge difference induced by the mutation. Therefore, Hb variants carrying a mutation without charge variation may be difficult to separate from normal hemoglobins using the above mentioned techniques [3–5]. The development of reversed-phase liquid chromatography (RP-LC) has enhanced the separation between normal and mutated globin chains from some variants with no charge difference, but with a change in hydrophobicity [6]. Nevertheless, in some cases, the detection of an abnormal hemoglobin cannot be performed by these techniques and then, more sophisticated methods are required [7]. In addition, classical electrophoretic and chromatographic methods may be unable to distinguish one variant from another among the nearly 1000 variants reported [4]. Furthermore, even if DNA analysis gives information about nucleotide sequence changes, it cannot provide information at the protein level particularly in the detection of any posttranslational modification (PTM). Mass spectrometry (MS) has been used for analysis of Hb variants for the last three decades. The pioneer work of Wada et al. in 1981 [8] on the analysis of tryptic peptides of Hb by MS has been rapidly followed by the development of globin chain and peptide analysis using electrospray ionization MS (ESI-MS) and matrix-assisted laser desorption ionization MS (MALDI-MS) [9,10]. In ESI, proteins or peptides are sprayed through a smalldiameter needle in an acidic aqueous/organic solution (water/ methanol or water/acetonitrile). A high voltage is applied to this needle to produce an aerosol from a Taylor cone. These droplets have positive charges due to protons from the acidic solution. Droplet evaporation leads to the formation of smaller droplets from which ions are desorbed. ESI generates multi-charged ions: [M + nH]n+, where the n value depends on the size of the protein/peptide and on the number of basic amino acid residues which are present. MALDI is performed by bombarding sample with a laser beam (usually a UV nitrogen laser, 337 nm). Sample is mixed with a UV adsorbent matrix. This matrix is a UV-absorbing organic compound used to facilitate vaporization and ionization. Protein or peptide solution is mixed to matrix solution on a target probe and the mixture is allowed to dry. The matrix

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compound crystallizes and protein/peptide molecules are included into the matrix crystals. Pulses of laser light lead to complex photochemical and chemical reactions of the matrix bringing about the protonation and desorption of protein/ peptide molecules. Ions formed by MALDI are mainly singlycharged ions [M + H]+. Mass analysis consists in the determination of the mass-tocharge ratio (m/z), of produced ions. A variety of mass analyzers are available to perform these measurements: quadrupole mass filter, ion trap, linear quadrupole trap, time-of-flight (TOF), Fourier transform-ion cyclotron resonance (FT-ICR) and orbitrap [9,10]. Sensitivity (ion transmission), resolution and mass range are the main characteristics of a mass analyzer. The resolution (R) is defined as (m/z)/(Δm/z) where Δm may represent either the mass difference between two ions or the difference between peakwidth at half-height. Mass spectra can be recorded at low resolution (1000 N R N 2000) using ion trap, quadrupole mass filter or linear trap analyzers. High resolution (5000 N R N 10,000) can be performed using TOF or quadrupole TOF analyzers. Higher resolution (10,000 N R N 100,000) requires orbitrap or Fourier transform-ion cyclotron resonance analyzers [9,10]. Low resolution MS of proteins cannot resolve the isotope cluster of the molecular ions and leads to broad peaks corresponding to the average mass of all isotopic contributions. High resolution MS of peptides/proteins allows to resolve the components of the isotope cluster and leads to the measurement of the monoisotopic mass or exact mass. This mass is obtained by considering only the lightest isotopes of the different elements [11,12]. This review reports the different MS techniques commonly used for the identification and the characterization of an Hb variant. It also shows the importance of the combination of DNA and MS studies in the detection of PTM, double mutations and complex mutations involving insertions and deletions. Limitations of MS methods will be also discussed. MS methods in Hb variant analysis Molecular weight determination of globin chains Sample preparation for ESI-MS and MALDI-MS analysis For ESI-MS analysis, a whole blood sample is diluted in a mixture of water/organic solvent (acetonitrile or methanol) and organic acid (acetic or formic acid) (49.9/49.9/0.2, respectively). The mixture is desalted by using cation-exchange resin beads (AG 50W-X8, BioRad Laboratories, Hercules, CA, USA)

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[13]. The globin fractions collected by RP-LC can also be used for MS analysis. For MALDI-MS analysis, blood samples are generally diluted and different matrixes can be used: ferulic acid, 2,6-dihydroxyacetophenone [14]. MS analysis Molecular weight determination provides crucial information about amino acid residues modifications. Since the development of ESI-MS and MALDI-MS, the analysis of the intact globin chain has been allowed [8,11]. The ESI spectrum

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of an intact human globin is rather complex (Fig. 1a). From this spectrum, it is possible, by using two successive peaks [M + nH]n+ and [M + (n + 1)H](n + 1)+, to obtain the molecular mass M. A mathematical process called deconvolution performs these calculations from the mass spectrometer data (Fig. 1b). In practice, ESI-MS analyses of globin chains are made at low resolution using ESI-quadrupole instruments, affording to average masse measurements with standard deviation (SD) values ranged from ± 0.5 to ± 2 Da. Using the same instrumentation, some particular analyses can be

Fig. 1. (a) ESI-MS of the multi-charged ions [M + nH]n+ derived from the α- and β-globin chains before deconvolution. Multi-charged ions derived from the α-globin chains; : multi-charged ions derived from the β-globin chains and M: molecular mass. (b) ESI-MS of the whole globin after deconvolution. Average masses of the αand β-globin chains are presented. Some minor components are detected.

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performed at a very low mass range acquisition using internal standards and maximum entropy-based software (MaxEnt® ) for deconvolution of multi-charged ions, in these cases, SDs b ± 0.05 Da are obtained [15]. The same performance can be made by MALDI-MS concerning mass accuracy. SDs of ± 4 Da are routinely obtained for proteins ranged between 15 and 20 kDa. Moreover, instruments operating at 1500–1700 resolution and using internal standards, a SD b ±0.3 Da is obtained for α- and β-globin chains determination [14]. The presence of a globin chain variant in a mixture can be detected with great confidence when the molecular mass differs by more than 6 Da from the normal α- and β-globin chains [12,13]. Nevertheless, high accuracies of mass measurements can be obtained in particular conditions as described above. Hence, it is possible to detect Hb variants that differ in mass from Hb normal by ± 1 Da such as Glu ↔ Lys, Glu ↔ Gln, Asp ↔ Asn, Asn ↔ Ile [15]. The direct analysis of intact globin chains by electrospray tandem MS (MS/MS) is not routinely applied for Hb variants characterization. The main interest of this method is to avoid labor-intensive preparations and proteolytic cleavage steps to obtain information about the amino acid sequence. Globin chain MS/MS studies have been carried out on different analyzers: ion trap [16], quadrupole TOF [17] and triple quadrupole [18]. But, MS/MS spectra are not easy to interpret and the main protein sequence information is only obtained from the C- and Ntermini regions [19]. This approach could be better using Fourier transform analyzers. MS analysis of globin chain-derived peptides Sample preparation An enzymatic digestion of globins is performed to produce peptides which are easier to characterize by MS techniques. Digestion can be done directly on the blood hemolysate or on the RP-LC purified globin sample. The reduction/alkylation steps are optional and depend on the tryptic peptides involved in the mutation (see section Problems in MS). The most used matrix for peptide MALDI analyses is the αcyano-4-hydroxycinnamic acid. Desalting and removal of organic compounds are carried out on micro-tips with C18 reversed-phase support [20]. The sample and the matrix solution are mixed on the target well-plate and allowed to dry (dry droplet method). For ESI tandem MS analysis, the peptide separation is performed in a capillary [16,21] or a nano-LC C18 reversedphase column [20]. Sample solutions are usually desalted by using an online C18 pre-column. MS analysis of globin chain-derived peptides A complete characterization of a Hb variant can be obtained by analysis of peptides produced by an enzymatic digestion of the globin chains. Only peptide masses ranging between 600 and 3000 Da are normally detected and can provide significant MS sequence information. Trypsin is the most commonly used enzyme for cleaving globin chains. It cleaves the peptidic bonds at the C-terminal side of lysine and arginine residues. However,

some globin tryptic peptides are too small to be characterized by MS as mentioned above. In order to get better sequence coverage other proteolytic enzymes such as chymotrypsin, endoprotease Glu-C, endoprotease Asp-N, have been used. Moreover, uncleaved peptides, longer than the previous ones, can be generated by using moderate proteolytic digestions. Two different MS approaches can be used for globin peptides analysis either peptide mass fingerprinting (PMF) which gives exact mass determination of proteolytic peptides or peptide sequencing by MS/MS instrumentation. An exact mass (monoisotopic mass) determination of the majority of globin tryptic peptides can be obtained. Changes in the amino acid sequence of the globin chain can be directly identified by the difference of the observed masses between the mutated and the normal tryptic peptides. Some rare mutations with no mass difference such as Leu → Ile or Gln → Lys are undetectable. The obtained information must be correlated to the DNA sequence analysis for confirming the mutation. Tandem MS (MS/MS) technique consists in the selection of specific peptide ions, followed by a fragmentation process in a collision cell and finally by a fragmented ions analysis. Collision-induced dissociation (CID) or collision-activated dissociation (CAD) experiments consists in ion acceleration in the presence of a collision gas. Typical instruments using CAD/ CID fragmentation are: triple quadrupole, quadrupole-TOF and TOF–TOF analyzers [9,10]. Another process for fragmenting ions is radio-frequency excitation, especially in ion trap and FTICR mass analyzers [9,10]. The ions obtained from CID fragment process of the peptide bond cleavage are conventionally named as y-ions series (from C-terminal of the peptide) and b-ions series (from N-terminal of the peptide). These ions are characteristic of the amino acid sequence of the peptide [19]. Then, localization and identification of the amino acid mutation can be accurately determined. In general, MS/MS techniques are associated with online separation by RP-LC in ESI or offline MALDI analysis. Screening methods using MS/MS of globin tryptic peptides An interesting approach for rapid detection of known Hb variants using LC-MS/MS has been recently developed and combines the use of a peptide database [21]. In this analytical method, Hb samples are digested by trypsin and the resulting peptide mixture is analyzed by LC coupled to tandem MS/MS. Experimental MS data are compared with a Hb variant peptide database derived from HbVar [4]. Another approach to determine known sequence modifications of peptides in MS is the multiple reaction monitoring (MRM), which is a very sensitive and specific detection and/or quantification for targeted analytes. MRM experiments require triple quadrupole mass analyzers instruments [10]. A typical MRM strategy starts by using the first quadrupole (Q1) to select a limited number of peptide ions obtained from the ESI with pre-specified m/z values (Fig. 2). Then, the fragmentation only targets the specific selected ion in the collision cell (Q2) with the optimized collision energy. The produced fragment ions are monitored on the third quadrupole (Q3) only for a specific pre-

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Oxidation of cysteinyl residues into cysteic acid by performic acid/hydrogen peroxide is another strategy to prevent disulfide bond formation [29]. Furthermore, the use of formic acid and hydrogen peroxide (H2O2) is an improved method to analyze core peptides from hemoglobin [30]. More recently, a mild oxidation procedure using a 7% H2O2 solution at room temperature for 15 min in the dark for vacuole proteomic studies has been described [31,32]. The main advantage of this oxidation procedure is to reduce sample preparation compared to the classical reduction/alkylation procedures. It was recently used to characterize a thalassemic α-chain variant (Hemoglobin Groene Hart) [20]. Hemoglobin variants with a single mutation in the globin chain Fig. 2. Multiple reaction monitoring (MRM) of some tryptic peptides derived from βA- and βS-globin chains using a triple quadrupole capability of a mass spectrometer. In a first mass analyzer (Q1) are selected the precursor ions of βT1 peptide with m/z 476.8 and m/z 461.8, corresponding to the doubly-charged ions [M + 2H]2+ of the normal βT-1 (M = 952.5 Da) of the βA-globin chain and the mutated βT-1 of the βS-globin chain (M = 922.5 Da), respectively. These peptides are fragmented into the collision cell (Q2) and selected into a second mass spectrometer (Q3) as y4 daughter ions and detected. Daughter ions studies of these precursor ions have shown that they produced a characteristic and intense y4 ion (from the C-terminal fragment ion with the proline effect in position 5). The detection for Q3 is fixed on these y4 fragment ions at m/z 502.3 and m/z 472.3 for the normal and the mutated βT-1, respectively.

identified daughter ion associated with a given precursor. A detailed example is presented by Daniel et al. [22] on MRM detection for Hb S in prenatal diagnosis analyses. Problems in MS: cysteinyl residues Although no disulfide bond occurs in normal hemoglobin, trypsin digestion of globin chains leads to peptides with free cysteinyl residues in the following tryptic peptides: αT-12, βT10 and βT-12. These peptides could form disulfide peptides. To prevent intra- or inter-molecular disulfide bond(s) formation during sample preparation, a chemical modification of thiol residues is recommended [13]. MS analysis without cysteine derivatization has been made but cysteine containing peptides were difficult to characterize by MS/MS [23]. Several types of cysteine modifications have been used: alkylation (aminoethylation/carboxyamidomethylation) or oxidation. Aminoethylation of cysteine was first obtained by an ethylenimine reagent [24]. It is a very efficient reagent with high reaction yield. However, being now listed as a carcinogen, it is no longer commercially available. It has been replaced by the 2-bromoethylamine [25] and more recently by the N-(βiodoethyl)trifluoracetamide (Aminoethyl-8™ reagent from Pierce) [26]. Piotrowki et al. [27] have shown that aminoethylation is more efficient with ethylenimine than with the two other reagents for proteins. Because aminoethylcysteine is isosteric with lysine, proteases like trypsin could cleave alkylated globin chains at this residue, leading to shorter tryptic peptides. Another widespread alkylation method used in proteomic sample preparation is iodoacetamide which provides carboxyamidomethylation of cysteine residues [28].

Several Hb variants with a single mutation in the globin chain have been detected and identified by using the combination of electrophoretic, chromatographic and mass spectrometric methods [4]. Clinical consequences of these mutations are very variable. Many Hb variants are clinically silent, but others induce from a mild to severe hemolytic anemia and for others an erythrocytosis or a thalassemic phenotype with hypochromia and microcytosis. For most Hb variants, ESI-MS applied to whole globin analysis gives the molecular mass of the mutated globin chain. MALDI-MS after tryptic digestion shows PMF of the abnormal globin chain. NanoLC-ESI-MS/MS determines the amino acid sequence of the mutated peptide(s). MS studies of large tryptic peptides is possible such as αT-9 peptide, the one of the two larger tryptic peptides (with the αT12 peptide) derived from the α-globin chain with a m/z ratio of about ∼ 3000. For example, two mutations located in αT-9: Hb Villeurbanne α89His → Tyr [33] and Hb Al-Hammadi Ryadh (α75Asp → Val) [34] were identified by MS methods including tandem MS/MS to sequence the mutated peptide. In a recent study [12], Kleinert et al. reported the results on a 5-year study including 2105 blood samples analyzed with cation-exchange LC (CE-LC), RP-LC and followed by ESIor MALDI-MS analysis for measurement of their globin chain masses. They found 4 samples with an Hb variant chromatographically silent which were only identified by MS. These Hb variants were Hb Riccarton (α51Gly → Ser) (2 unrelated patients) and two new Hb variants: Hb ZurichHottingen (α9Asn → Ser) and Hb Zurich-Langstrasse (β50Thr → Ser). These Hb variants induced no clinical consequences. The authors argued (on the basis of the Hb variants listed in HbVar and according to all possible single mutations) that two-thirds of the mutations with a possible charge difference had been previously reported. By contrast, minus 30% of the variants without any charge difference had been previously described. Another point emphasized by Kleinert et al., was the limitation of ESI-MS analysis using conventional mass spectrometers for separating globin chains with a mass difference of less than 6 Da. However, the use of MALDI-MS on the tryptic peptides can allow to draw back this limitation. In addition, NanoLC-ESI-MS/MS allows improving the detection of Hb variants by an accurate analysis of their constitutive peptides.

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Hemoglobin variants with two amino acid substitutions in a single globin chain Several Hb variants with two amino acid substitutions in the same globin chain have been reported [4]. The detection of these substitutions in the same polypeptide chain by MS (ESI-MS or MALDI-MS) can be difficult because the observed mass shift is a cumulative sum of the respective shifts provided by each mutation. These variants result in a new mutation in a gene containing one mutation. For example, the worldwide mutation in hemoglobin S [β6Glu → Val] has been reported in association with other mutations in the same β-globin chain [4]. Clinical consequences of these cis associations are highly variable. Sickle cell disease has been observed at the heterozygous state, for example, in Hb SAntilles, or combined with Hb C [4]. At the opposite, some Hb variants are clinically asymptomatic such as Hb S-Clichy [35] or Hb S-Providence [4]. MS can also discriminate between a cis and trans association of a new mutation with βS mutation. For example, Hb Quebec-Chori (β 87Thr→ Ile), an electrophoretically silent Hb variant [36], which was associated with Hb S in trans, led to a severe form of sickle cell trait. Some other Hb variants with a double amino acid mutation but without βS mutation have been reported [4]. These mutations are also β-globin chain variants The first mutation often corresponds to a common mutation such as Hb C [β6Glu → Lys], Hb D-Punjab [β121Glu → Gln] or Hb E [β26Glu → Lys] [37]. But, some Hb variants do not have a common mutation, such as Hb Villeparisis [β77His → Tyr, β80Asn → Ser], a clinically silent Hb variant, which was detected during a measurement of glycated hemoglobin [38]. This variant was identified by ESI-MS on the tryptic digest of the β-globin chain and tandem MS/MS of the mutated βT-9 peptide. Another Hb variant also found during a routine HbA1c measurement was Hb Kochi [39]. MS studies of tryptic peptides of Hb Kochi demonstrated the presence of a double mutation in the same β-globin chain: 141 Leu → Val and 144 Lys → 0, leading to an increase in oxygen affinity. These results were confirmed by DNA analysis. Hemoglobin variants with an insertion or a deletion in a globin chain Some Hb variants can reveal either an elongated globin chain or a shortened globin chain. Depending on the type and the

location of the amino acid residues inserted or deleted in the αor β-globin chain, clinical consequences are variable. Insertion can be clinically silent like in Hb Zaire where an insertion of five amino acid residues occurs between α116 and α117 [40]. By contrast, insertion can induce severe instability of the Hb molecule with hemolytic anemia or mimic a thalassemic phenotype such as in Hb Koriyama which presents an insertion of five residues between β95 and β96 [40]. In addition, functional properties of Hb can be impaired such as in Hb Catonsville with an insertion of Glu residue between α37 and α38 [40]. Interpretation of DNA studies in the cases of insertion or deletion of nucleotides can be difficult at the heterozygous state. Consequently, it is important to study the amino acid sequence of the mutated globin chain. Some Hb variants with an elongated or deleted chain have been studied using MS methods and are presented in Table 1. Hemoglobin variants with post-translational modifications Some Hb variants present a PTM that cannot be detected by DNA analysis and then, MS studies are required. These PTM have been ranged in three categories: (i) PTM on the Nterminal globin chain; (ii) PTM involving deamidation of asparagine residue into aspartate residue; (iii) others PTM associated with more complex changes in the polypeptide chain. PTM linked to the non-enzymatic addition of glucose on the N-terminal valine residue of the β-globin chain and known for giving glycated Hb A1c and the same glucose addition on the N-terminal valine of the α-globin chain and ɛgroups of the lysine residues of the α- and β-globin chains are excluded from this review. Hb variants reported with PTM are presented in Table 2. Unstable hemoglobin variants Common laboratory diagnosis of unstable Hb variants includes Heinz bodies detection, routine biochemical methods, and heat and isopropanol stability tests [67]. Stability tests can allow to purify the unstable variant. ESI-MS analysis on the whole lysate and the precipitate obtained from isopropanol or heat stability test can detect the mutated globin chain. ESI- or MALDI-MS of tryptic peptides confirms the mutation. This strategy has been applied to the identification and

Table 1 Some Hb variants with inserted or deleted amino acid residues and studied by MS techniques. Hb variant name

Inserted/deleted amino acid sequence

Position of the insertion/deletion

Globin chain mass difference (Da) a

Reference

Hb Esch Hb Epsom

Ala-Leu-Thr-Asn Ala-Leu-Ser-Ala-Leu-Ser-Asp-LeuHis-Ala-His-Lys-Leu-Arg-Val Arg-Val-Leu-Ala-His Gly-Ser-Ala-Gln-Val-Lys-Gly-His → 0 or: Ser-Ala-Gln-Val-Lys-Gly-His-Gly → 0

Between α1 68(E17) and 69(E18) Between α2 94(G1) and 95(G2)

+400 +1614

[41] [42]

Between β115(G17) and β117(G19) Between α1 51(CE9) and 58(E7) or: Between α1 52(E1) and 59(E8)

+439.29 − 765.5

[43] [44]

Hb Antibes-Juan-Les-Pins Hb J-Biskra a

Measured by ESI-MS. Mass difference relative to the normal α- or β-globin chain mass.

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Table 2 Hemoglobin variants reported with post-translational modifications. N-terminal α- or β-globin chain

Deamidation of Asn into Asp

Other PTM

Hb Thionville: α1Val → Glu with α Met-Glu and α N-Ac Met-Glu [45] Hb Antananarivo: α1Val → N-Ac-Gly [46] Hb Lyon Bron: α1Val → N-Ac-Val [47]

Hb Hb J-Singapore: α78 Asn → Asp and α79 Ala → Gly [54,55] Hb J-Sardegna: α50 His → Asn → Asp [56] Hb Wayne: α139-141 Lys-Tyr-Arg → Asn-Thr-Val-Lys-Leu-Glu-Pro-Arg → Asp-Thr-Val-Lys-Leu-Glu-Pro-Arg [57,58] Hb Providence: β82 Lys → Asn → Asp [59]

Hb Atlanta-Coventry: β 75Leu → Pro with β 141Leu → OHLeu [63,64] Hb Bristol: β 67Val → Met → Asp [65,66]

Hb Saint Josef: α1Val → Leu with α Met-Leu and N-Ac Met-Leu [48] Hb Doha: β1Val → Glu with β Met-Glu and N-X Met-Glu [49] Hb Marseille: β 2His → Pro with β Met-Val [50] Hb South Florida: β1Val → Met with βMet-Met and β N-Ac Met-Met [51] Hb Niigata: β1Val → Leu with β Met-Leu and N-Ac Met-Leu [52] Hb Raleigh: β1Val → N-Ac-Val [53]

Hb Osler: β145 Tyr → Asn → Asp [60] Hb Redondo: β92His → Asn → Asp [61] Hb la Roche-sur-Yon: β81 Leu → His and β80Asn → Asp [62] Hb Sapporo: β143His → Asn → Asp [4]

characterization of some unstable Hb variants [4]. The observed mass variation by ESI-MS can be indicative of a point mutation with a limited number of possibilities according to the genetic code. In Hb Canterbury [β112 (G14)Cys → Phe], the normal and mutated β-globin chains were detected in ESI-MS as adducts with glutathione [4]. PMF on tryptic digest of the precipitated globin confirmed the mutation. Some mildly unstable Hb variants can present a β+or α+-thalassemia-like expression. For example, Hb Mont Saint Aignan [β12(H6)Ala → Pro], the variant was isolated by selective precipitation with isopropanol and structural analysis performed by RP-LC with MS [68]. Fusion hemoglobin variants Hb Lepore is a class of Hb variants with normal α-globin chains and abnormal non-α globin chains which are hybrid proteins [4]. These hybrid proteins contain the N-terminal sequence of a δ chain and the C-terminal sequence of a β chain. The classical mechanism for Hb Lepore formation results from different recombination events between the δ- and β-globin genes and consequently the presence of a (δβ)0-deletion. Four types of Hb Lepore have been described so far [4,69]. Their differences in structure correspond to the variation of position for the transition from δ chain to β chain. Hb Lepore-Boston (δ87-β116) and Hb Lepore-Baltimore (δ50-β86) are the most frequent encountered. Hb Lepore-Hollandia (δ22-β50) is a rare Hb variant. Recently described Hb Lepore-Leiden is a fusion Hb with complex protein structure resulting from crossover recombination associated with gene conversion [69]. Hb Lepore is a low-expressed variant, at the heterozygous state, it accounts for 5% to 15% of the total hemoglobin. Compound heterozygotes with β-thalassemia trait can give a thalassemia intermedia phenotype or with hemoglobin S a mild sickle cell disease. Biochemical diagnosis is performed by routine electrophoresis, CE-LC, RP-LC and DNA studies (see reference 69 for a recent review). ESI-MS can easily measure molecular masses of globin chains for common Hbs Lepore [70]. Structural identification of Lepore chain can be performed

by the analysis of tryptic digest by ESI- or MALDI-TOF-MS [70,18]. Another fusion Hb is Hb Kenya (Aγ81Leu-β86Ala). This variant is characterized by an abnormal non-α chain, a hybrid A γβ chain also resulting from crossing-over of Aγ- and βgenes within a gene loci [4]. Exact diagnosis of Hb Kenya can be difficult and requires DNA analysis combining gap-PCR and DNA sequencing [71]. ESI-MS can help to diagnose the presence of the A γβ chain [72]. Hemoglobin variants with low expression Some Hb variants are low expressed and associated with a thalassemic phenotype with hypochromia and microcytosis. At the heterozygous state, these variants can often be associated with borderline hematological parameters. Recent studies have focused on α-chain Hb variants with an α-thalassemia (α-thal) phenotype. These variants can be unstable and/or present defects in binding with α-hemoglobin stabilizing protein (AHSP) [73]. An interesting example of this type of α-chain Hb variants is Hb Groene Hart [α119 (H2) Pro → Ser, (α1)]. This variant is detected by RP-LC of whole hemolysate because the αGroene Hart-globin chain is highly hydrophobic [20]. The variant at the heterozygous state is low expressed and hematological parameters show slight hypochromia and microcytosis. However, at the homozygous or compound heterozygous state with an α+-thal gene (-α3.7), the variant percentage is increased and identification and characterization has been carried out by semi-preparative RP-LC followed by MS studies. It is interesting to emphasize that in some α-thal Hb variants, no protein can be detected by LC and MS such as Hb Foggia [α117(GH5)Phe → Ser (α2)] [73,4]; Hb Voreppe [α123(H6)Ala → Pro (α1))] [73,4] or Hb Tunis-Bizerte [α129 (H12)Leu → Pro (α1)] [73,4]. Hemoglobin variants giving high molecular weight species In deconvolution processing of data in ESI-MS, Hb globin chains are usually selected as monomeric species but, in some

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cases of Hb variants, a complex mixture of monomers, dimers and high molecular weight species is present during ESI-MS analysis and can complicate identification. The problem must be considered in the case of the replacement of an amino acid residue by a cysteinyl residue. In HbVar (updated on January 31, 2008), a strictly limited number of Hb variants with a substitution leading to a cysteinyl residue is reported: 5 α-chain variants and 12 β-chain variants [4]. In some Hb variants, the new inserted cysteinyl residue can form intrachain and/or interchain disulfide (-S–S-) bonds. In the case of Hb Porto Alegre (β9Ser → Cys), the cysteinyl residue is located in the external part of the Hb molecule and interchain disulfide bonds are formed [4]. Hb Porto Alegre was studied by chromatographic techniques combining RP-LC and gel filtration chromatography and ESI-MS in native and denatured conditions [74]. It was demonstrated that Hb Porto Alegre is able to give an assembly of dimers (about 30%) and high molecular weight oligomers with covalent bonds. These species were found to have functional properties. In the case of Hb Harrow [β118Phe → Cys], ESI-MS studies showed that a small percentage of dimer of βHarrow was formed [75]. In the case of Hb Ta-Li [β83Gly → Cys], ESI-MS studies proved that the variant also existed as a dimeric species with an interchain disulfide bridge [76]. In the case of Hb Rainier (β145Ser → Cys), a high oxygen affinity Hb variant, an intramolecular disulfide bond appeared between β145Cys with the internal β 93 normal cysteinyl residue [4]. By contrast, in Hb Nunobiki [α141Arg→Cys], no interchain and/or intrachain disulfide bond was detected [4].

Sardinia. In ESI-MS, the masses of Gγ and AγT chains are separated by only 2 Da and cannot be differentiated [77]. In neonatal blood samples, acetylation of the N-terminal extremities of the G γ- and Aγ-globin chains occurs because N-terminal glycyl residue does not inhibit N-terminal acetyltransferases [81]. Acetylation is easily detected in ESI-MS by a +42 Da mass increase [77]. The embryonic ɛ- and ζ-globin chains are easily separated from G γ- and A γ-globin chains by the RP-LC method previously described [20]. Embryonic ζ-globin chains show a strong increase of hydrophobicity in RP-LC with a complete acetylation of the N-terminal seryl residue that can be detected only by ESI-MS. The presence of persistent embryonic ζ-globin chains have been described in some carriers of deletions leading to some types of α-thalassemia [82]. MS and neonatal diagnosis of hemoglobinopathies Neonatal screening of hemoglobinopathies for the detection of sickle cell disorders is performed in many countries by using CE-LC and isoelectric focusing (IEF) [83]. Blood samples are transmitted as dried spots called “Guthrie cards” and hemoglobin analysis is performed after elution. ESI-MS and MALDITOF-MS have been applied to the screening of hemoglobinopathies in some laboratories [84–86]. Recently, F. Boemer et al. [86] have developed a newborn screening for sickle cell disease using tandem MS. A minimal treatment of the Guthrie card spots followed by a tryptic digestion has provided a working solution for tandem MS/MS analysis using a MRM mode of specific peptides analysis.

Embryonic and fetal hemoglobins Minor components: Hb A2 and delta-chain variants In a normal neonate blood sample, two main types of fetal hemoglobin are observed with a point change in the γ-globin chain in position 136: Gγ (136Gly) and Aγ (136Ala). In ESIMS, these two globin chains are discriminated with their distinct molecular masses. From ESI-MS of a fetal blood sample, it can be interesting to use the relative abundances corresponding to the different heights of the peaks of γ-globin chains for estimating the Gγ/Aγ ratios [77]. MS techniques can be extended to the structural characterization and identification of variants of γ-globin chains [4]. These variants are often discovered during the neonatal screening for hemoglobinopathies. Most of these variants have no clinical consequences. However, among the 59 mutations of the Gγ-globin gene [4] (HbVar updated on January 31, 2008), a limited number have various abnormal properties such as: instability [2]; high oxygen affinity [2] or methemoglobinemia formation [2]. Among the 50 mutations of the Aγglobin genes variants which have been described, none show functional abnormalities. MS has been used for the identification of some γ-chain variants, for example: Hb F-Bron [Aγ20 (B2)Val → Ala] [78], Hb F-Clamart [Gγ17(A4)Lys → Asn] and Hb F-Ouled Rabah [Gγ19(B1)Asn→Lys] [79]. MS has also allowed to demonstrate a N-terminal acetylation of a γ-chain variant [80]. A common variant of the Aγ-globin chain is the mutation A γ 75Ile → Thr which is the fetal globin chain of Hb F-

Hb A2 is a normal and minor component of hemoglobin and comprises 2 α- and 2 δ-globin chains [87]. In adult blood samples, Hb A2 in normal red blood cells is expressed at low concentrations (2–3% of total Hb). Hb A2 concentrations are generally increased (4–6%) in carriers of a β-thalassemic trait [86]. Hb A2 concentrations are estimated by automated CE-LC in routine Hb analysis and also in the screening of βthalassemia carriers. In a recent study, Daniel et al. [88] have presented a new method based on tandem MS to compare the proportion of δ- and β-globin chains in normal subjects and carriers of a β-thalassemia trait. An important pitfall is that the δ-globin chain differs from β-globin chain by only 10 amino acid residues [87]. Comparisons of the δ/β peptide ratios in MS/MS analysis using the MRM mode have been performed for some informative peptides of the two globin chains. In another study, Zurbriggen et al. [89] have studied minor hemoglobins including Hb A2 after chromatographic separation by CE-LC followed by MALDI-TOF MS of the different globin chains. About 80 δ-globin chain variants have been described so far. These variants can present normal properties but also give a δ0or δ+-thalassemia [4]. The low expression of Hb A2 and the δglobin chain variants induces little clinical consequences. The identification of δ-globin chain variant is performed by DNA

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studies. The confirmation by structural analysis requires variant isolation by ion-exchange LC and after trypsin digestion can be performed by NanoLC-MS/MS such as the case of Hb Noah Mehmet Oeztuerk [90]. Methemoglobins Biochemical diagnosis of methemoglobins (Hbs M) includes electrophoretic and chromatographic Hb methods combined to spectrophotometric recording in the visible spectrum [91]. DNA analysis can confirm the mutation and MS studies can easily verify the amino acid sequence of the mutated globin chain. Few cases of Hb M have been structurally characterized by using MS techniques. Hb M Hyde Park [β92 (F8) His → Tyr] [92,93] and Hb M Iwate [α87(F8) His → Tyr] [33]. In that last study, the mutation concerned the large α-T9 peptide. The characterization of the structural abnormality required ESI-MS of abnormal globins and α-T9 peptides associated with tandem MS/MS of the corresponding tryptic peptides. Perspectives MS has become an often used technique for Hb analysis. It has first been a complementary technique to confirm globin variant identification. A true screening method by triple quadrupole analysis using MRM strategy has been developed and could replace the classical CE-LC and IEF analytical methods. However, these screening MS methods are dedicated to blood samples containing known hemoglobin variants. The development of MS applied to the proteomic field has allowed the analysis of globin variants and has been used for the study of the cytoplasmic proteome of the human erythrocytes [94]. Like many proteomic studies, the bottom-up MS strategy which starts from digested peptides can resolve most of the characterization of Hb mutation, despite of time consuming on sample preparation. The other possibility is the top-down MS strategy, i.e. MS studies of intact proteins. At that step, only the molecular weight of the intact globin chains is measured, but direct sequencing of the intact protein is also available by FTICR instruments [95]. In that case, the efficient protein sequencing by tandem MS is obtained by electron capture dissociation (ECD) [96]. This technique is able to fragment proteins using thermal electrons excitation [96] instead of collision energy techniques (CID/CAD). Nevertheless, FT-ICR remains the most expensive and powerful MS technique. An alternative method would be the use of electron transfer dissociation (ETD) in the linear ion trap, where anions are the electron provider to multiple charged peptides or proteins [97]. In this experiment anions are produced by negative chemical ionization. ETD induces ion fragmentations that are analogous to those observed in ECD (c- and z- type fragment ions). This technology allows protein sequencing [97], especially if the fragment ions are analyzed in an orbitrap mass analyzer with high mass accuracies and charged states determination. Moreover, bioinformatics software developments will help MS data interpretation.

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Conclusions Increase of MS performance and developments in the proteomic fields have made a great impact on hemoglobin variant analysis. These performances can be applied at different steps of the globin variant analysis process: either as a screening method or as an additional technique to confirm the results from classical analytical methods. In a classic issue, a mass difference above 6 Da will be necessary to differentiate whether a mutation is present or not. This limitation in MS determination is due to the complexity between the normal and mutated globin chains. This limitation is overcome by using high resolution instruments (FT-ICR, Orbitrap) or by special precautions on low resolution instruments. For these low mass differences between normal and variant globin chains, MS analysis of digested peptides is required. At the peptide level, after enzymatic digestion, all types of variants can be characterized, but in some cases, like Hb Cairo β65 Lys → Gln, high resolution is needed to differentiate Lys from Gln (0.036 Da mass difference). In addition to this, DNA sequencing data, which are very complementary to the MS analysis, will help the MS/MS interpretation. In our experience, the combination of three classical analytical techniques: IEF, CE-LC and RP-LC can detect almost all Hb variants. However, the use of MS techniques can detect unusual Hb variants which are silent in these analytical techniques. Moreover, MS is the only technique which allows characterizing PTM of globins. In conclusion, Hb variant analysis by MS has been strongly intensified during the last two decades which is quoted by the number of publications dedicated to this topic. References [1] Wajcman H, Prehu C, Bardakdjian-Michau J, et al. Abnormal hemoglobins: laboratory methods. Hemoglobin 2001;25:169–81. [2] Riou J, Godart C, Hurtrel D, et al. Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants. Clin Chem 1997;43: 34–9. [3] Hardison RC, Chui DHK, Giardine B, et al. Hb Var: a relational database of human hemoglobin variants and thalassemia mutations at the globin gene server. Hum Mutat 2002;19:225–33. [4] HbVar: a Database of Human Hemoglobin Variants and Thalassemias. http://globin.cse.psu.edu/hbvar/menu.html (Accessed January 31, 2009). [5] Giardine B, Van Baal S, Kaimakis P, et al. HbVar database of human hemoglobin variants and thalassemia mutations: 2007 update. Hum Mutat 2007;28:206. [6] Leone L, Monteleone M, Gabutti V, Amione C. Reversed-phase highperformance liquid chromatography of human haemoglobin chains. J Chromatogr 1985;321:407–19. [7] Troxler H, Neuheiser F, Kleinert P, et al. Detection of a novel variant human hemoglobin by electrospray ionization mass spectrometry. Clin Chem 2002;292:1044–7. [8] Wada Y, Hayashi A, Fujita T, Matsuo T, Katakuse I, Matsuda H. Structural analysis of human hemoglobin variants with field desorption mass spectrometry. Biochim Biophys Acta 1981;667:233–41. [9] Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature 2003;422:198–207. [10] Domon B, Aebersold R. Mass spectrometry and protein analysis. Science 2006;312:212–7. [11] Wada Y. Advanced analytical methods for hemoglobin variants. J Chromatogr, B 2002;781:291–301.

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