Quantification of Hemoglobin A2 by Tandem Mass Spectrometry

Abstract

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 delta:beta-globin peptide ratios as potential surrogate markers of HbA(2), a biomarker used in population screening for beta-thalassemia trait. We studied 163 blood samples with normal HbA(2) (%), 105 with increased HbA(2), 43 with delta-chain variants, and 8 with Hb Lepore. All were tested by HPLC. The samples were also incubated with trypsin for 30 min at 37 degrees C for MSMS with flow injection analysis. MRMs for the delta- (T2, T3, and T14) and beta- (T2, T3, and T13) globin tryptic peptides were acquired for 1 min, and delta:beta 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. Within- and between-assay imprecision values (CVs) were <6.1% and <8.4%, respectively, for the delta:beta peptide ratios. Digests were stable at 10 degrees C for 6 days. Significant correlations (P <0.0001) between MSMS delta:beta-globin peptide ratios and HPLC HbA(2) allowed differentiation between increased HbA(2) 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. This study validates the quantitative delta:beta-globin peptide ratio as a surrogate marker of HbA(2) and demonstrates the potential of rapid peptide-based MSMS for multiplexed, high-throughput protein biomarker characterization and quantification.