Hemoglobin Wayne in a British Family: Identification by Electrospray Ionization/Mass Spectrometry

Hemoglobin (Hb) Wayne (an elongated α-chain frameshift variant that exists as 2 forms, Hb Wayne Asn and Hb Wayne Asp) was first described in 1976 in a Caucasian neonate in Pike County, Central Georgia, whose grandparents originated from Birmingham, Alabama (1). Two other families have been reported, one a Caucasian family from Michigan (2), and the other from Canada (3). Clinically, Hb Wayne leads to an increased oxygen affinity Hb with noncooperativity and a markedly reduced Bohr effect. Hb Wayne Asn and Hb Wayne Asp exhibit differences in their carbon monoxide-binding properties (4). We report the identification of Hb Wayne in a 28-year- old Caucasian diabetic female patient whose blood was undergoing analysis for glyco-Hb. Her initial pathologic investigations showed no abnormalities (144 g/L Hb; erythrocytes, 5.09 × 1012/L; packed cell volume, 0.437 L/L; mean cell volume, 85.8 fL; mean cell Hb, 28.3 pg; mean cell Hb concentration, 330 g/L). Analysis of a hemolysate for glyco-Hb using a HA-8140 analyzer (Menarini Diagnostics Ltd.) revealed a slight shoulder on the glycated Hb peak with an estimated HbA1c concentration of 13.8% [Diabetes Control and Complications Trial (DCCT)-aligned]. This analyzer uses HPLC on a methylacrylic acid/methylacrylate ester column (HSV-II-V column; Sekisui Chemical Co Ltd.) with a sequence of three eluants producing a HbA1c result every 4 min. Other common hemoglobins (HbA2, HbC, HbF, HbS, and metHb) were excluded because their retention times on the analyzer are characteristic and different from that of the unusual peak. A sample was referred for electrospray ionization/mass spectrometry [(electrospray ionization/MS); Ref. (5)]. The mass spectrometrically estimated HbA1c was 61 g/L. Electrospray ionization/MS analysis of intact α- and β-chains (5) using a Micromass Quattro Ultima tandem mass spectrometer showed that the abnormal Hb contained an α-chain with a mass (15617.54 ± 0.05 Da; n = 4) 491.16 Da heavier than the normal chain (Fig. 1), suggesting the variant Hb Wayne. Tandem MS (MS/MS) analysis of the intact variant and normal chains showed that the altered sequence lay at the C-terminal end of the α-chain and gave supporting evidence for Hb Wayne. Final confirmation of the sequence was obtained from a tryptic digest, followed by MS/MS of the variant peptides (αT12–13 and αT14). Electrospray mass spectrum of blood from the patient (1:500 dilution). A second α-chain peak can be seen, 491.1 Da heavier than the normal α-chain. All of these results were fully consistent with the sequence of Hb Wayne except the mass of the variant α-chain. This was substantially higher (0.60 Da) than predicted from the sequence of Hb Wayne (15616.94 Da), but would occur if there were partial (60%) deamidation of Asn139 to Asp139. Such partial deamidation has already been described for this variant (2)(3)(4), in which the proportions of the Asn139 and Asp139 species were approximately equal. MS analysis cannot resolve these species because their mass difference is only 1 Da and measures their abundance weighted mean mass. Hence, the predicted mass for the combined species, assuming equal proportions, is 0.50 Da higher than the sequence mass of the Asn139 species and is within experimental error of the measured mass. Therefore, all the mass spectral data were consistent with the Hb Wayne frameshift mutation, leading to replacement of the three C-terminal amino acids, α139–141, by Asnα139-Thr-Val-Lys-Leu-Glu-Pro-Arg146 together with partial deamidation of Asn139 (40%) to Asp139 (60%). The patient was heterozygous for the variant, which was present at 18% of total α-chains. The previously reported cases of Hb Wayne were identified by biochemical methods. In electrospray ionization/MS, ions are formed from intact polypeptides by adding one or more protons (H) (5). MS measures the mass to charge ratio (m/z) of these ions. Because the number of charges (n) is known, the molecular mass (Mr) in daltons (Da) is deduced using Mr = n(m/z − H), and software converts the original m/z data into a mass spectrum as in Fig. 1 of the data supplement (available with the online version of this Technical Brief at http://www.clinchem.org/content/vol48/issue12/. In effect, MS/MS combines the purification and sequencing of polypeptide mixtures, previously undertaken by the use of several procedures into one operation without chromatography. The technique uses an instrument consisting essentially of two mass spectrometers in series. The first spectrometer purifies the polypeptide mixture by selecting ions with a specific m/z. These ions are then fragmented by collisions with a gas in a cell situated between the two spectrometers. The resulting fragment ions are then analyzed according to their m/z in the second spectrometer to give sequence information. Mainly, fragment ions result from cleavages between the amino acid residues in the polypeptide chain. The skill in MS lies in identifying diagnostic peaks in complex spectra with the aid of computer programs. The complete MS analysis of the variant Hb was carried out and results were returned to the referring laboratory within 4 working days, a substantial reduction compared with biochemical techniques. This is the third abnormal Hb we have reported that has been analyzed by this technique (6)(7)(8), and we recommend it as a rapid method for identification of unknown variants, although we caution that interpretation requires considerable expertise.

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