High pressure liquid chromatography and electrospray ionization mass spectrometry are advantageously integrated into a two-levels approach to detection and identification of haemoglobin variants

Abstract

Detecting and correctly identifying haemoglobin (Hb) variants is typically achieved by a two-levels laboratory approach. We report our experience in dealing with 91 Hb variants, including a number of frequent and a few rare variants. Screening included akaline agarose gel electrophoresis (AGE), ion-exchange automated high-performance liquid chromatography (HPLC) and a test for deoxyhaemoglobin solubility. Identification was based on electrospray ionization-mass spectrometry (ESI-MS). Our results confirmed the advantages of HPLC over AGE for screening, because of the occurrence of some electrophoretically 'silent' variants. ESI-MS permitted the definitive identification of 90 of the 91 variants included in the study, in some cases (e.g. HbS) through the application of a simple protocol (direct injection of the sample), in other cases requiring the application of more demanding procedures (purification of the variant chain and peptide analysis after enzymatic or chemical cleavage). In an additional case (Hb J-Oxford), ESI-MS assay did not lead to definitive identification, but gave indications for designing the appropriate primers to focus DNA sequence analysis on the specific region of the gene. Deoxyhaemoglobin solubility test was positive only in the presence of HbS. We conclude that HPLC and ESI-MS are advantageously integrated into a two-level analytical system for the detection and confirmation of variant Hbs.