Rapid Identification of Hemoglobin Variants by Electrospray Ionization Mass Spectrometry

Detection and quantitation of normal and variant haemoglobins: an analytical review

“…These analytical procedures will detect the majority of the common clinically important haemoglobin variants and enable a presumptive identi¢ca-tion of the variant to be made, which is often su⁄cient for clinical purposes. In order to identify the variant unequivocally it is necessary to analyse either the protein structure by the use of mass spectrometry, 21 which has largely replaced ¢ngerprinting with amino acid analysis, or to perform DNA analysis using gene sequencing or speci¢c restriction enzyme techniques. 22,23 A summary of the analytical procedures employed in the various clinical situations is given in Box 2.…”

Characterization of a Double Heterozygote HbE/β+ Thalassemia IVS 1-1 [G>T] in a Juvenile Diabetic

“…Of course, although ESI-MS of the globin chains can detect ±1 Da mutations, precise identification requires enzymatic digestion. Hemoglobins C, D-Punjab, E, and O-Arab can all be positively identified directly from the mass spectra of 30 min digests with trypsin, which was reconfirmed by Wild BJ et al [11]. Rai et al have mentioned about the usage of ESI-MS in the study of hemoglobin variants, whereas the method adapted here is unique, as it involves characterizing hemoglobin variant, which is obtained through the steps of performing alkaline gel electrophoresis, excising and proteolytically digesting the hemoglobin gel bands to obtain peptides; and finally analyzing the peptides by MALDI-MS.…”

Characterization of the elusive disulfide bridge forming human Hb variant: Hb Ta-Li β83 (EF7)Gly → Cys by electrospray mass spectrometry

“…A stock solution of 50-fold diluted whole blood was prepared in water. A mass spectrometric sample was made by 10-fold dilution of the stock solution with solvent A (1:1 acetonitrile:water containing 0.2% formic acid) and desalted as previously described [4]. The sample was introduced into the ES ion source at a flow rate of 8 L/min.…”

“…M ass spectrometry has become widely used in the investigation of Hb variants since it was first used by Wada et al as a novel method for the identification of variant Hb in 1981 [1][2][3][4][5][6][7][8][9][10]. Electrospray (ES) in combination with tandem mass spectrometry (MS/MS) is a commonly used technique for the identification of Hb variants [2][3][4][5][6][7][8][9][10].…”

“…Electrospray (ES) in combination with tandem mass spectrometry (MS/MS) is a commonly used technique for the identification of Hb variants [2][3][4][5][6][7][8][9][10]. Shackleton and Witkowska [3] and more recently Wild et al [4] have elaborated the application of ES mass spectrometry to the identification of Hb variants. In our laboratory ES mass spectrometry is used as a secondary as well as a final tool in the identification process following screening of the blood samples by HPLC and/or isoelectric focusing [5,6].…”