Hemoglobin debrousse (β96[FG3]Leu → Pro): A new unstable hemoglobin with twofold increased oxygen affinity

Lacan, Philippe; Kister, J.; Francina, Alain; Souillet, G; Galactéros, Frédéric; Delaunay, Jean; Wajcman, Henri

doi:10.1002/(sici)1096-8652(199604)51:4<276::aid-ajh5>3.0.co;2-t

Cited by 40 publications

(16 citation statements)

References 13 publications

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“…1 and 2 with the MS/MS sequencing results of two characteristic peptides from the ␤ and ␦ globin parts, respectively. On the ␤-part of the fusion hemoglobin, three specific amino acid sequences [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], [18][19][20][21][22][23][24][25][26][27][28][29][30] and were determined: The MS/MS spectrum of the amino acid sequence was presented as the entire sequence of the ␤-part of the fusion hemoglobin. The detailed results are shown in Table 1 (LC-MS/MS chromatogram and ion detection are reported in Supplementary data).…”

Section: Resultsmentioning

confidence: 99%

“…EDTA blood samples from three unrelated patients were collected for a complete screening of hemoglobinopathies: CE-LC analyses were carried out on Variant II (Bio-Rad, Hercules, CA, USA) using the 'Short Beta Thal program'; IEF on polyacrylamide gels was performed as previously described [3]; CEP analysis used a Sebia Capillarys apparatus (Sebia, Lisses, France) and reversed-phase liquid chromatography of globin chains (RP-LC) was performed on a column C 4 Uptisphere (4.6 mm × 250 mm, 5 m particle, average pore size 300Å, Interchim, Montluç on, France) as previously described [4].…”

Section: Protein Analysismentioning

confidence: 99%

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Protein characterization by LC–MS/MS may be required for the DNA identification of a fusion hemoglobin: The example of Hb P-Nilotic

Zanella-Cléon

Delolme

Lacan

et al. 2012

Journal of Chromatography B

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Section: Resultsmentioning

confidence: 99%

Section: Protein Analysismentioning

confidence: 99%

Protein characterization by LC–MS/MS may be required for the DNA identification of a fusion hemoglobin: The example of Hb P-Nilotic

Zanella-Cléon

Delolme

Lacan

et al. 2012

Journal of Chromatography B

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“…Blood samples were drawn into collection tubes containing EDTA, and hematologic data were collected via standard methods. Hemolysates were analyzed by isoelectric focusing on polyacrylamide gels (11,12 ), ion-exchange LC (Variant I™, Beta-Thalassemia Short Program; Bio-Rad Laboratories) (11,12 ), and RPLC of globin chains.…”

Section: Patients and Hematologic Proceduresmentioning

confidence: 99%

“…Genomic DNA was extracted from whole blood as previously described (11,12 ). The sequence of the Hb Groene Hart mutation was identified via PCR of the HBA1 7 (hemoglobin, alpha 1) and HBA2 (hemoglobin, alpha 2) genes and direct DNA sequencing of the PCR products on a LI-COR 4200 sequencer (LI-COR Biosciences).…”

Section: Dna Analysismentioning

confidence: 99%

Detection of a Thalassemic α-Chain Variant (Hemoglobin Groene Hart) by Reversed-Phase Liquid Chromatography

et al. 2008

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BACKGROUND: Hemoglobin (Hb) Groene Hart [␣119 (H2)Pro3 Ser (␣1)], also known as Hb Bernalda, is a nondeletional ␣-thalassemic Hb variant that is frequent in southern Italy and North Africa. This variant is not supposed to be produced in the erythrocytes of carriers. The ␣-thalassemic behavior of this variant has been explained as an impaired interaction between the ␣-globin chain and the ␣-Hb-stabilizing protein.

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“…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 C 4 analytical column (The Separations Group) with CH 3 CN-H 2 O containing 1 mL/L trifluoroacetic acid (TFA) as the mobile phase (7 ).…”

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Identification by Mass Spectrometry of a Hemoglobin Variant with an Elongated β-Globin Chain

et al. 2005

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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 (NH 2 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 ␤-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 C 4 analytical column (The Separations Group) with CH 3 CN-H 2 O 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 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 ϳ5 mg of AG 50W-X8 cation-exchange resin (Bio-Rad). We diluted 20 L of the desalted stock solution by adding 40 L of CH 3 OH-H 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 ϩ H] ϩ ions. For MALDI-TOF sample preparation, we dried 0.5 mL of the 2-mL ␤-globin chain sample collected from RP-HPLC purification in a vacuum concentrator and redissolved the residue in 90 L of 50 mmol/L NH 4 HCO 3 buffer. This solution was digested with 10 L of trypsin solution (Promega; 0.1 g/L in 50 mmol/L NH 4 HCO 3 buffer) for 5 h at 37°C. We then dried 10 L of the digest so...

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Hemoglobin debrousse (β96[FG3]Leu → Pro): A new unstable hemoglobin with twofold increased oxygen affinity

Cited by 40 publications

References 13 publications

Protein characterization by LC–MS/MS may be required for the DNA identification of a fusion hemoglobin: The example of Hb P-Nilotic

Protein characterization by LC–MS/MS may be required for the DNA identification of a fusion hemoglobin: The example of Hb P-Nilotic

Detection of a Thalassemic α-Chain Variant (Hemoglobin Groene Hart) by Reversed-Phase Liquid Chromatography

Identification by Mass Spectrometry of a Hemoglobin Variant with an Elongated β-Globin Chain

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