MSACL 2022 Abstract

Introduction:
Hemoglobinopathies are heritable diseases related to disorders of hemoglobin (Hb). Normal adult Hb (Hb A) is a tetramer with two α and two β subunits. Two other normal adult Hbs are Hb A2 (α2δ2, no more than 3.5%) and Hb F (α2γ2, no more than 1%). Thalassemia syndrome, one type of hemoglobinopathy, is an endemic disease affecting approximately 7% of the world population and is characterized by a reduced production of α or β subunits, resulting in α-thalassemia or β-thalassemia. In β-thalassemia, the two genes (β/β) can vary from deficit (β+) to defective (β0), so there are three sub-types including β-thalassemia trait (β0/β or β+/β), intermedia (β0/β or β+/β+) and major (β0/β0 or β+/β0). Patients with β-thalassemia major and intermedia are easier to identify because β-thalassemia major patients often fail to thrive as infants and intermedia often causes significantly different clinical symptoms including jaundice, cholelithiasis, organ lesion, etc.. On the contrary, β-thalassemia trait carriers are often asymptomatic with decreased amount of Hb A and elevated Hb A2, Hb F, so the relative amount of Hb A2 to the other normal hemoglobins is critical to the identification of β-thalassemia trait.

Objectives:
HPLC-FT-ICR-MS with/without proton transfer reaction- parallel ion parking (PTR-PIP) was used to relative quantify intact Hb subunits, and δ/β ratio as a biomarker can distinguish β-thalassemia with optimal cutoff points.

Methods:
The intact Hb subunits were separated by high-performance liquid chromatography to reduce the complexity and characterized as intact by 21 T FT-ICR MS. The relative quantitation based on the delta-to-beta (δ/β) ratio from area under the curve mass spectral peaks is used to distinguish β-thalassemia and non-β-thalassemia samples. To further increase the sensitivity of detection, PTR-PIP, in which the radical anion reagent abstracted protons from multiply charged precursor ions and the produced charge-reduced ions are parked at defined m/z range, was applied to increase the S/N ratio of the subunits of interest. The Mann-Whitney U test was used to determine whether there is a difference in the δ/β ratio for the two groups. Receiver operating characteristic (ROC) curve and Youden criteria were used to determine the diagnostic performance of diagnosis method and obtain the optimal cutoff points.

Results:
The box-and-whisker and ROC plots illustrate that δ/β ratio can serve as a biomarker to distinguish β-thalassemia in normal MS1 at a cutoff point (optimal threshold value) of 2.61% with a sensitivity (the probability of a true positive) of 100% and a specificity (the probability of a true negative) of 90%, and in PTR-PIP MS1 at a cutoff point of 2.92% with a sensitivity of 100% and a specificity of 100%. The charge states of precursor ions are concentrated into fewer charge states (usually one or two) in PTR-PIP MS1 experiments; therefore not only did the S/N ratios of the Hb subunits increase but also provided improved sensitivity and specificity.

Conclusion:
We demonstrate that δ/β ratio calculated from HPLC-MS experiments can serve as a biomarker to identify β-thalassemia, and the PTR-PIP technique improves the sensitivity and specificity. The number of samples (14 for β-thalassemia and 21 for not β-thalassemia) suffices to test the practicability of our method, but larger sample size is needed to obtain a clear cutoff point with fewer false-positive results.

A portion of this work was performed at the National High Magnetic Field Laboratory ICR User Facility, supported by the National Science Foundation Division of Chemistry through Cooperative Agreement No. DMR-1644779 and the State of Florida.