= Emerging. More than 5 years before clinical availability. (19.79%)
= Expected to be clinically available in 1 to 4 years. (37.97%)
= Clinically available now. (42.25%)
MSACL 2022 : Lin

MSACL 2022 Abstract

Self-Classified Topic Area(s): Emerging Technologies > Various OTHER > none

Podium Presentation in De Anza 2 on Thursday at 16:30 (Chair: Timothy Collier)

Discovery of a Biomarker for beta-Thalassemia by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Proton Transfer Reaction- Parallel Ion Parking

Yuan Lin(1), Archana M. Agarwal(3,4), Lissa C Anderson(2), Alan G. Marshall(1,2)
(1) Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32308 (2) Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310 (3) University of Utah, School of Medicine, Salt Lake City, UT 84132 (4) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108

Yuan Lin (Presenter)
Florida State University

Presenter Bio: I am a fifth-year Ph.D. candidate in Dr.Alan Marshall's group at Florida State University. My research area is related to applying top-down tandem mass spectrometry to study intact proteins/proteoforms using 21 T Fourier transform ion cyclotron resonance mass spectrometry at the National High Magnetic Field Laboratory.
Research includes identification of hemoglobin and beta-thalassemia by HPLC top-down MS/MS with CID/ETD and new techniques such as chimeric ion loading and proton transfer reaction (PTR) - parallel ion parking, and the improvement of the fragmentation effeminacy such as ETD.


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.

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.

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.

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.

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.

Financial Disclosure

GrantsyesThe National High Magnetic Field Laboratory, Ion Cyclotron Resonance Facility
Board Memberno
IP Royaltyno

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