Abstract

Identification of Single Mutations in Hemoglobin Variants Using Modified Sample Preparation and Digestion Method and Separation Power of LC Coupled with MALDI MS/MS

Suman Kundu, Professor, University of Delhi

Around 7% of the global population carries an abnormal hemoglobin gene. Over 330,000 infants are born annually with hemoglobinopathies and it is the major cause of morbidity and mortality in early childhood. The treatments rely heavily on the diagnosis of hemoglobin variants. The routine/conventional techniques used for the identification of mutation in hemoglobin variants have their own limitations like co-migration of variants in electrophoresis and co-elution in HPLC. The WHO (2002) report on Genomics and Health has emphasized on the development of precise molecular techniques for screening of hemoglobin disorders. A sensitive, robust and reproducible method was thus developed to identify single substitution mutations in the hemoglobin disorders from sequence of the entire globin chains. The method was MS compatible and dealt with certain limitations like difficulty in getting complete sequence coverage. Using a specific organic solvent, digestion with a combination of proteases, treating the digestion mixture with 10% acetonitrile prior to incubation and combining the separation power of LC coupled with MALDI MS/MS resulted in 100% sequence coverage in the ß chains and very high sequence coverage in the a chains. A hemoglobin variant database was created to specify the search and reduce the search time. All the mutations were thus identified using a non-targeted approach. This method could be used in future for regular screening of any single mutation in hemoglobin variants.