Hb Neuilly-sur-Marne, a new human hemoglobin variant with Ser-Asp-Leu inserted between alpha 86(F7) Leu and alpha 87(F8) His: characterization by high-energy collision-induced dissociation liquid secondary ion mass spectrometry and low energy collision-induced dissociation tandem mass spectrometry in an ion trap fitted with a nanospray ionization source

Hemoglobin (Hb) Neuilly-sur-Marne is a new a-chain variant found during a s ystematic screening. Electrospray mass measurements showed the presence of an abnormal alpha -chain displaying a shift of +315 u relative to the norma l value. Tryptic cleavage of this chain and molecular weight determination of the peptides indicated that the 315 u shift was located into the alphaT- 9 peptide, the molecular weight of which is higher than 3000 Da. High-energ y collision spectra of MH+ ions generated by liquid secondary ion mass spec trometry from the normal and abnormal alphaT-9 afforded mainly amino-termin al containing ions. They indicated that these two peptides have an identica l amino acid sequence from their 1st to 25th residues, the mass increase be ing thus located beyond this point. Too few ions were formed to establish r eliably the sequence forward. It was hypothesized that this mass shift coul d result from a repeated sequence since the sum of the mass of the three re sidues-leucine, serine and aspartic acid-preceding position 25 is exactly 3 15 u. To get sequence information above position 25, decomposition of multi charged species was attempted. An ion trap fitted with a nanospray ionizati on source was used. It produced mainly triply- and quadruply-charged ions. Decomposition of the triply-charged ion afforded a series of singly-charged Y-ions in the expected region, giving a readily interpretable sequence. It confirmed the insertion of a Ser-Asp-Leu sequence above position 25. Surpr isingly, decomposition of the quadruply-charged molecular ion gave too few ions to provide sequence information in the expected region. Spectra were d ominated by some multicharged Y ions arising from cleavages close to the am ino end. Tandem mass spectrometry experiments were performed on the abundan t Y-30(3+) ion and produced again a singly-charged Y ion series in the suit able domain which confirmed the above result. In Hb Neuilly-sur Marne this insertion of the Ser-Asp-Leu residues, between positions alpha -86 and alph a -87 is very likely due to a slipped strand mispairing mechanism.