New Results of Hemoglobin Variant Structure Determinats by Fast Atom Bombardment Mass Spectrometry

Abstract

Fast atom bombardment mass spectrometry has already been used for the identification of mutations in abnormal human hemoglobin chains. This paper presents new results obtained with this technique. The methodology used here is compared with more conventional biochemical techniques and automated microsequencing. In every case, a well-chosen combination of peptide-high performance liquid chromatography, mass spectrometry, amino acid analysis, and sequence analysis led rapidly to the identification of the mutant. The high sensitivity of these techniques holds great promise for the analysis of molecular abnormalities in various genetic disorders presently detectable only by the application of a molecular biological approach.

Detection and characterization of variant and modified structures of proteins in blood and tissues by mass spectrometry

“…They used field desorption MS using a special emitter, that is, very fine uniform silicon whiskers (Matsuo, Matsuda, & Katakuse, 1979) and obtained peptide maps of normal and variant Hbs. Later, methods for Hb variant analysis using fast atom bombardment (FAB) ionization or liquid matrix in secondary ion MS (L‐SIMS) were developed (Lacombe et al, 1990; Lee & Rahbar, 1991; Wada et al, 1992a). Those pioneering studies were followed by work using modern soft ionization (Falick et al, 1990; Green et al, 1990; Covey, Huang, & Henion, 1991; Shackleton & Witkowska, 1996).…”

Hemoglobin Phnom Penh [α117Phe(H1)-Ile-α118Thr(H2)]; evidence for a hotspot for insertion of residues in the third exon of the α1-globin gene

“…Electrospray mass spectrometry measurements were done as previously described (Lacombe et al, 1990).…”

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

“…From the eleventh to the thirteenth position, the ions that contain the Assuming a single amino acid substitution, a 1-u shift downward corresponded to either Glu -> Gin, Asp -> Asn, or Asn -> He exchange. Another allowed exchange was Glu -> Lys, but because lysine is a cleavage site for trypsin, this proposal very likely has to be ruled out [11]. None of these amino acid exchanges was consistent with the electrophoretic behavior of the variant.…”

“…Assuming that the point mutation is due to a single base substitution in a cod on triplet of the DNA, all the amino acid exchange possibilities are not allowed. According to the genetic code, there is a strong limitation in the number of possible amino acid exchanges that are compatible with a measured mass shift [11,12].…”