Use of combined mass spectrometry methods for the characterization of a new variant of human hemoglobin: The double mutant hemoglobin villeparisis β77(EF1) His → Tyr, β 80 (EF4) Asn → Ser

Abstract

Hemoglobin Villeparisis was found during a systematic measurement of glycated hemoglobin. Electrospray mass spectra of the globin indicate an apparently unchanged molecular weight within the error range (0.01%). The tryptic digest of the β chain shows a chromatographically abnormal βT-9 peptide. The mass-to-charge ratio value of its [M+H](+) ion, as measured by liquid secondary ionization mass spectrometry, is one mass unit lower than that of the normal βT-9. However, the electrospray mass spectrum of this peptide exhibits mainly a doubly charged ion, whereas the normal βT-9 gives a triply charged ion. None of the allowed single amino acid substitutions for a 1-u shift down (Glu → Gln, Asp → Asn, or Asn → Ile) can explain the suppression of one protonation site. This can be due only to the replacement of the internal histidine by a nonbasic residue. Thus at least two amino acid exchanges occur within the same peptide: one involves the internal histidine, and the sum of the mass shifts is -1 u. Consideration of the βT-9 sequence and taking account for the genetic code rules, the only possibility was (11)His → Tyr (+26 mass shift) associated with (14)Asn → Ser (-27 mass shift). This conclusion was consistent with the tandem mass spectrum of the [M+H](+) ion and was further confirmed by chemical microsequencing.

Ion Trap Collisional Activation of the (M + 2H)2+ − (M + 17H)17+ Ions of Human Hemoglobin β-Chain

“…In analogy with the work already performed to evaluate techniques to coax structural information from protein ions in the FTICR environment, − methods for dissociating protein ions in the quadrupole ion trap environment must be evaluated to optimize the extent of structural information that can be obtained with this form of instrumentation. Human hemoglobin β-chain represents a protein of obvious clinical interest, − the tandem mass spectrometry of which is not straightforward with conventional benchtop tandem mass spectrometers due to difficulties in interpreting the spectra. In this work, we explore the dissociation behavior of hemoglobin β-chain ions over a wide range of charge states using conventional ion trap single-frequency collisional activation .…”

Strategy for Identification by Mass Spectrometry of a New Human Hemoglobin Variant with Two Mutations inCisin the β-Globin Chain: Hb S-Clichy [β6(A3)Glu→Val; β8(A5)Lys→Thr]

“…As in the case of Hb S-Clichy, the mass difference which does not fit with a single amino acid exchange suggests a complex structural modification requiring further studies. In other cases, as in Hb Villeparisis (35), the mass differences resulting from the two substitutions will compensate, and ESI-MS will fail to demonstrate the presence of the two abnormalities. It will require MALDI-TOF or MS/MS studies of tryptic peptides to decipher the sequence modification.…”

Abnormal Hemoglobins: Laboratory Methods

“…Nevertheless, several limitations to the method need to be mentioned: a too small difference in mass will not lead to a signi®cant displacement of the signal, and a too small amount of abnormal component may give a signal dif®cult (or impossible) to recognize from the background noise. Some dif®culties in the interpretation of the data may also result from complex abnormalities, such as the presence of more than one point mutations, a deletion or an insertion of residue(s) (23). The possibility for covalent dimers also needs to be considered: in the case of Hb Porto Alegre [b9(A6)Ser 3 Cys] we observed that the abnormal b chain was totally in the form of dimers covalently bound through an S-S bridge.…”