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Identification by mass spectrometry of a hemoglobin variant with an elongated [beta]-globin chain.

Among more than 800 hemoglobin (Hb) variants currently described in the HBVar database of the Globin Gene Server (1), variants with elongated chains are very rare. Standard protein techniques such as ion-exchange HPLC and isoelectric focusing (IEF) on polyacrylamide gel can detect many Hb variants (2), but correct identification of single mutated, inserted, or deleted amino acid residues requires more sophisticated techniques, such as electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (3-5). The interpretation of DNA sequencing in the presence of inserted nucleotide sequences in the heterozygous state can be difficult and requires direct and reverse sequencing. Protein analysis by MS can be used to check results from DNA sequencing and also can detect posttranslational changes such as acetylation (NHZ terminus), deamidation, methionine oxidation, Hb addition products, or artifacts. ESI-MS and MALDI-TOF MS combined with specific tryptic digestion for peptide mass mapping are rapid and sensitive techniques to confirm inserted amino acid residues. We applied these techniques to the identification of a novel Hb variant with a five-amino acid insertion in the [beta]-globin chain. These techniques allowed us to confirm the DNA sequencing results.

Standard Hb analysis was performed by ion-exchange HPLC, IEF on polyacrylamide gel, and reversed-phase (RP)-HPLC of globin chains (6,7). RP-HPLC was performed with a Vydac C4 analytical column (The Separations Group) with CH3CN-H20 containing 1 mL/L trifluoroacetic acid (TFA) as the mobile phase (7). The same RP-HPLC system was also used for isolation of globin chains for MS studies. Electrospray experiments were performed on a SCIEX API 165 instrument (Applied Biosystems). Solutions were introduced by direct infusion at a flow rate of 5 [micro]L/min. Mass spectra were acquired at a 50-V orifice value in the positive ion mode, and the scan range was set at m/z 650-2150 Th (Thompson).

For ESI-MS samples, we prepared a stock solution by diluting the whole blood 50-fold with water and desalting the stock solution by mixing with ~5 mg of AG 50W-X8 cation-exchange resin (Bio-Rad). We diluted 20 [micro]L of the desalted stock solution by adding 40 [micro]L of C[H.sub.3]OH-[H.sub.2]O (50:50 by volume) containing 1 mL/L HCOOH, and this solution was used for ESI-MS analysis. MALDI-TOF mass spectra were recorded on a Voyager DE-PRO mass spectrometer (Applied Biosystems) in the 700-5000 Da mass range. Use of a delayed extraction source and reflector instrumentation gave sufficient resolution to detect the monoisotopic peptide masses of [[M + H].sup.+] ions.

For MALDI-TOF sample preparation, we dried 0.5 mL of the 2-mL [beta]-globin chain sample collected from RP-HPLC purification in a vacuum concentrator and redissolved the residue in 90 [micro]L of 50 mmol/L N[H.sub.4]HC[O.sub.3] buffer. This solution was digested with 10 [micro]L of trypsin solution (Promega; 0.1 g/L in 50 mmol/L N[H.sub.4]HC[O.sub.3] buffer) for 5 h at 37[degrees]C. We then dried 10 [micro]L of the digest solution in a vacuum concentrator and redissolved it in 10 [micro]L of 1 mL/L TFA. The matrix was a 1 mg (200 [micro]L) solution of a-cyano-4-hydroxycinnamic acid (LaserBioLabs) in C[H.sub.3]CN-[H.sub.2]O (50:50 by volume) containing 1 mL/L TFA. We used the dried-droplet method for sample deposition: 1 [micro]L of the 1 mL/L TFA sample solution and 1 [micro]L of matrix solution were mixed on the target and allowed to dry.

DNA studies were carried out by PCR using the following primers: 5'-CAGCTACAATCCAGCTACCATTCTGCT-3' (forward) and 5'-TAGGCAGAATCCAGATGCTCAAGGCCC-3' (reverse) (7). Direct sequencing of the PCR products was performed on a Li-Cor 4200 sequencer (ScienceTec) using the same primers labeled with an infrared dye with emission at 700 run (forward strand) or an infrared dye with emission at 800 run (reverse strand; MWG-Biotech). Functional studies were made on a Hemox-Analyzer (TCS Scientific Corporation) after stripping of blood hemolysate.

Routine protein analysis showed an abnormal Hb band in the IEF gel that migrated between Hb S and Hb [A.sub.2]; this band accounted for 22.6% of the total Hb in ion-exchange HPLC. RP-HPLC analysis of the globin chains detected an abnormal peak 20.62 min after the [[beta].sup.A]-globin chains (Fig. 1A). ESI-MS analysis revealed a mass of 16307.0 Da (([[beta].sup.x]) vs 15868.0 for the [[beta].sup.A]-globin chain (mass shift, +439.0). DNA sequencing revealed an insertion of a short nucleotide sequence, GTGTGCTGGCCC, in exon 3 of the [beta]-globin gene. The same sequence was also found in exon 3 of the wild-type [beta]-globin gene between the second nucleotide of codon 112 and the first nucleotide of codon 116 (Table 1). We hypothesized that [beta]116(G18)His was deleted and the peptide sequence Arg-Val-Leu-Ala-His was inserted between 0115(G17) and [beta]117(G19) of the normal ([beta]-globin chain (Table 1). MALDI-TOF MS was performed on a tryptic digest of the [[beta].sup.X]-globin chain to confirm this interpretation. The [[beta].sup.X]-chain should contain 150 amino acid residues vs 146 for the normal [[beta].sup.A] chain with a theoretical mass shift of +439.29. Theoretical masses of tryptic peptides from [[beta].sup.X]- and [[beta].sup.A]-chains (8) were compared with the experimentally obtained peptide masses (Table 1).

The experimentally obtained masses of different peptides were very close to their theoretical masses. The two tryptic peptides, [beta]T12 and [beta]T13, generated by the five-amino acid insertion were characterized by MALDI-TOF MS (Fig. 1B). The present Hb variant was called Hb Antibes-Juan-Les-Pins and was associated with hematologic abnormalities: erythrocytes, 5.41 x [10.sup.3] /L; Hb, 137 g/L; mean corpuscular volume, 78 fL; and mean corpuscular Hb, 25.3 pg. The variant was also found in two children of the same carrier. They presented with the same hematologic abnormalities without iron deficiency. Oxygen dissociation curves performed after stripping with or without the addition of 2,3-diphosphoglycerate or chloride ions were normal.

Among MS techniques for studying Hb variants, ESI-MS is the most frequently used and can be associated with peptide sequencing using tandem MS, but it often gives multiply charged fragment ions (4,5). On the other hand, MALDI-TOF MS gives single-charge peptide ions and has been used for identification of some single-mutation Hb variants (9,10). The present report shows that MALDI-TOF MS can be use to identify Hb variants. This MALDI-TOF peptide mass fingerprinting method is currently used in our laboratory for abnormal globin chains analysis.

The presence of the 12-nucleotide repeat strongly suggests that the origin of this insertion is probably based on a slipped mispairing by DNA polymerase during replication. Such an explanation has been proposed for different Hb variants (11). Amino acid residues at positions [beta]115-[beta]119 at the end of helix G in the normal [beta]-globin chain are involved either in [alpha]1[beta]1 subunit links or externally on the Hb molecule (12). The insertion of five amino acid residues leads to the addition of a complete helix turn that has no effect on oxygen binding but decreases molecule stability.


(1.) Hardison RC, Chui DH, Giardine B, Riemer C, Patrinos GP, Anagnou N, 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.

(2.) Wacjman H, Pr6hu C, Bardakjian-Michau J, Prom6 D, Riou J, Godart C, et al. Abnormal hemoglobins: laboratory methods. Hemoglobin 2000;25:169-81.

(3.) Shackleton CHL, Falick AM, Green BN, Witkowska HE. Electrospray mass spectrometry in the clinical diagnosis of variant hemoglobins. J Chromatogr B 1991;562:175-90.

(4.) Wild BJ, Green BN, Cooper EK, Lalloz RA, Erten S, Stephensen AD, et al. Rapid identification of hemoglobin variants by electrospray ionization mass spectrometry. Blood Cells Mol Dis 2001;27:691-704.

(5.) Wada Y. Advanced analytical methods for hemoglobin variants. J Chromatogr B 2002;781:291-301.

(6.) Lacan [MICRO] Kister J, Francina A, Souillet G, Galacteros F, Delaunay J, et al. Hemoglobin Debrousse [[beta]96(FG3)Leu [right arrow] Pro]: a new unstable hemoglobin with twofold increased oxygen affinity. Am J Hematol 1996;51:276-81.

(7.) Masala B, Manca L. Detection of globin chains by reversed-phase high-performance liquid chromatography. Methods Enzymol 1994;231:21-44.

(8.) Expasy Molecular Biology Server. Peptide mass. http: tools/peptide-mass.html (accessed August 2004).

(9.) McCombs ME, Oleschuk RD, Chow A, Ens W, Standing KG, Perreault H. Characterization of hemoglobin variants by MALDI-TOF MS using a polyurethane membrane as the sample support. Anal Chem 1998;70:5142-9.

(10.) Kiernan UA, Black JA, Williams [MICRO] Nelson RW. High-throughput analysis of hemoglobin from neonates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Clin Chem 2002;48:947-9.

(11.) Prehu C, Groff [MICRO] Kalmes G, Golinska B, Riou J, Prome D, et al. Short insertion in a hemoglobin chain: Hb Esch, an unstable a1 variant with duplication of the sequence [Ala.sup.65]-Leu-Thr-[Asn.sup.68]. Blood Cells Mol Dis 2003;31:234-9.

(12.) Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of hemoglobin: genetics, pathophysiology and clinical management. Cambridge: Cambridge University Press, 2001:174-96.

DOI: 10.1373/clinchem.2004.042630

Philippe Lacan, [1] Michel Becchi, [2] Isabelle Zanella-Cleon, [2] Martine Aubry, [1] Denis Quinsat, [3] Nicole Couprie, [4] and Alain Francina [1]

[1] Unite de Pathologie Moleculaire, Federation de Biochimie et de Biologie Specialisee, Hopital Edouard Herriot, Lyon, France; [2] Institut de Biologie et Chimie des Proteines (CNRS-UMR 5086), Lyon, France; [3] Laboratoire Marcel Merieux, Lyon, France; 4 Service de Medecine Interne, Centre Hospitalier, Antibes, France; * address correspondence to this author at: Unite de Pathologie Moleculaire, Federation de Biochimie et de Biologie Specialisee, Hopital Edouard Herriot, 69437 Lyon Cedex 03, France; fax 33-472110598, e-mail
Table 1. Codons, amino acid residues, and tryptic peptides from
[[beta].sup.X]-and [[beta].sub.A]-globin chains.

FROM [[beta].sub.X]-GLOBIN CHAIN (a)

105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121
 L L G N V L V C V L A R V L A H H

FROM [[beta].sub.X]-GLOBIN CHAIN

Position in
[[beta].sub.X]- Peptide Theoretical Experimental
chain name mass (b) mass (b) Sequence

 96-104 [beta]T11 1126.56 1126.57 LHVDPENFR
105-116 [beta]T12 1269.77 1269.76 LLGNVLVCVLAR
117-124 [beta]T13 908.51 908.50 VLAHHFGK
125-136 [beta]T14 1378.70 1378.70 EFTPPVQAAYQK
137-148 [beta]T15 1149.67 1149.68 VVAGVANALAHK
149-150 [beta]T16 319.14 YH

FROM [[beta].sub.A]-GLOBIN CHAIN (a)

105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
 L L G N V L V C V L A H H F G K

FROM [[beta].sub.A]-GLOBIN CHAIN

Position in
[[beta].sub.X]- Peptide Theoretical Experimental
chain name mass (b) mass (b) Sequence

 96-104 [beta]T11 1126.56 1126.57 LHVDPENFR
105-120 [beta]T12 1719.97 1719.98 LLGNVLVCVAHHFGK
121-132 [beta]T13 1378.70 1378.69 EFTPPVQAAYQK
133-144 [beta]T14 1149.67 1149.67 VVAGVANALAHK
145-146 [beta]T15 319.14 YH

(a) Italics, single-underlined, or double-underlined indicate
the inserted nucleotide sequence: GTGTGCTGGCCC.

(b) Expressed in mass-to-charge (m/z) ratio (monoisotopic mass).
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Title Annotation:Technical Briefs
Author:Lacan, Philippe; Becchi, Michel; Zanella-Cleon, Isabelle; Aubry, Martine; Quinsat, Denis; Couprie, N
Publication:Clinical Chemistry
Date:Jan 1, 2005
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