Quantification of Hemoglobin A2 by Tandem Mass Spectrometry

Abstract

Abstract Background: Peptide-based analysis of whole blood using electrospray tandem mass spectrometry (MSMS) in multiple reaction monitoring (MRM) mode enables rapid detection and sequence confirmation of clinically significant hemoglobin (Hb) variants. We applied a similar, quantitative approach to the measurement of δ:β-globin peptide ratios as potential surrogate markers of HbA2, a biomarker used in population screening for β-thalassemia trait. Methods: We studied 163 blood samples with normal HbA2 (%), 105 with increased HbA2, 43 with δ-chain variants, and 8 with Hb Lepore. All were tested by HPLC. The samples were also incubated with trypsin for 30 min at 37 °C for MSMS with flow injection analysis. MRMs for the δ- (T2, T3, and T14) and β- (T2, T3, and T13) globin tryptic peptides were acquired for 1 min, and δ:β peptide ratios were calculated. We used HPLC and MSMS to analyze 26 paired whole blood and dried blood spot samples after storage for 1, 8, and 29 days. Results: Within- and between-assay imprecision values (CVs) were <6.1% and <8.4%, respectively, for the δ:β peptide ratios. Digests were stable at 10 °C for 6 days. Significant correlations (P <0.0001) between MSMS δ:β-globin peptide ratios and HPLC HbA2 allowed differentiation between increased HbA2 concentrations and concentrations within the reference interval and identification of Hb Lepore. This differentiation was repeatable by MSMS, but not by HPLC, after blood spot samples had been stored for 1 month. Conclusion: This study validates the quantitative δ:β-globin peptide ratio as a surrogate marker of HbA2 and demonstrates the potential of rapid peptide-based MSMS for multiplexed, high-throughput protein biomarker characterization and quantification.