Discovery of a biomarker for β-Thalassemia by HPLC-MS and improvement from Proton Transfer Reaction - Parallel Ion Parking

Yuan Lin; Archana M Agarwal; Lissa C Anderson; Alan G Marshall

doi:10.1016/j.jmsacl.2023.01.004

Discovery of a biomarker for β-Thalassemia by HPLC-MS and improvement from Proton Transfer Reaction - Parallel Ion Parking

J Mass Spectrom Adv Clin Lab. 2023 Feb 2:28:20-26. doi: 10.1016/j.jmsacl.2023.01.004. eCollection 2023 Apr.

Authors

Yuan Lin¹, Archana M Agarwal^{2

3}, Lissa C Anderson⁴, Alan G Marshall^{1

4}

Affiliations

¹ Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32308, United States.
² Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132, United States.
³ ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, United States.
⁴ Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310, United States.

Abstract

β-thalassemia is a quantitative hemoglobin (Hb) disorder resulting in reduced production of Hb A and increased levels of Hb A₂. Diagnosis of β-thalassemia can be problematic when combined with other structural Hb variants, so that the separation approaches in routine clinical centers are not sufficiently decisive to obtain accurate results. Here, we separate the intact Hb subunits by high-performance liquid chromatography, followed by top-down tandem mass spectrometry of intact subunits to distinguish Hb variants. Proton transfer reaction-parallel ion parking (PTR-PIP), in which a radical anion removes protons from multiply charged precursor ions and produces charge-reduced ions spanning a limited m/z range, was used to increase the signal-to-noise ratio of the subunits of interest. We demonstrate that the δ/β ratio can act as a biomarker to identify β-thalassemia in normal electrospray ionization MS1 and PTR-PIP MS1. The application of PTR-PIP significantly increases the sensitivity and specificity of the HPLC-MS method to identify δ/β ratio as a thalassemia biomarker.

Keywords: ACN, Acetonitrile; AUC, Areas under the curve; CID, Collision-induced dissociation; ESI, Electrospray ionization; ETD, Electron-transfer dissociation; FA, Formic acid; FN, False-negative; FP, False-positive; FT-ICR; FT-ICR, MS Fourier transform ion cyclotron resonance mass spectrometer; FTMS; Fourier transform ion cyclotron resonance; Hb A, Normal adult Hb; Hb, Hemoglobin; HbA1d, Hb β with glutathione; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine; IQR, Interquartile range; J, Youden Index; MCW, Methanol/chloroform/water; MS, Mass spectrometry; PTM, Post-translational modification; PTR-PIP, Proton transfer reaction-parallel ion parking; ROC, Receiver operating characteristic; S/N, Signal-to-noise ratios; Se(c), Sensitivity, the probability of a true positive); Sp(c), Specificity, the probability of a true negative); TIC, Total ion chromatogram; TN, True negative; TP, True positive; Top-down; XIC, Extracted ion chromatograms; m/z, Mass-to-charge ratios; δ/β ratio.