Identification by Mass Spectrometry of a Hemoglobin Variant with an Elongated β-Globin Chain

Abstract

Among more than 800 hemoglobin (Hb) variants currently described in the HBVar database of the Globin Gene Server (1), variants with elongated chains are very rare. Standard protein techniques such as ion-exchange HPLC and isoelectric focusing (IEF) on polyacrylamide gel can detect many Hb variants (2), but correct identification of single mutated, inserted, or deleted amino acid residues requires more sophisticated techniques, such as electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (3)(4)(5). The interpretation of DNA sequencing in the presence of inserted nucleotide sequences in the heterozygous state can be difficult and requires direct and reverse sequencing. Protein analysis by MS can be used to check results from DNA sequencing and also can detect posttranslational changes such as acetylation (NH2 terminus), deamidation, methionine oxidation, Hb addition products, or artifacts. ESI-MS and MALDI-TOF MS combined with specific tryptic digestion for peptide mass mapping are rapid and sensitive techniques to confirm inserted amino acid residues. We applied these techniques to the identification of a novel Hb variant with a five-amino acid insertion in the β-globin chain. These techniques allowed us to confirm the DNA sequencing results. Standard Hb analysis was performed by ion-exchange HPLC, IEF on polyacrylamide gel, and reversed-phase (RP)-HPLC of globin chains (6)(7). RP-HPLC was performed with a Vydac C4 analytical column (The Separations Group) with CH3CN–H2O containing 1 mL/L trifluoroacetic acid (TFA) as the mobile phase (7 …