LC-MS/MS-Based Absolute Quantitation of Hemoglobin Subunits from Dried Blood Spots Reveals Novel Biomarkers for -Thalassemia Silent Carriers

Identification of alpha-thalassemia silent carriersis challengingwith conventional phenotype-based screening methods. A liquid chromatographytandem mass spectrometry (LC-MS/MS)-based approach may offernovel biomarkers to address this conundrum. In this study, we collecteddried blood spot samples from individuals with three alpha-thalassemiasubtypes for biomarker discovery and validation. We observed differentialexpression patterns of hemoglobin subunits among various alpha-thalassemiasubtypes and normal controls through proteomic profiling of 51 samplesin the discovery phase. Then, we developed and optimized a multiplereaction monitoring (MRM) assay to measure all detectable hemoglobinsubunits. The validation phase was conducted in a cohort of 462 samples.Among the measured hemoglobin subunits, subunit mu was significantlyupregulated in all the alpha-thalassemia groups with distinct foldchanges. The hemoglobin subunit mu exhibits great potential asa novel biomarker for alpha-thalassemia, especially for silent alpha-thalassemia.We constructed predictive models based on the concentrations of hemoglobinsubunits and their ratios to classify the various subtypes of alpha-thalassemia.In the binary classification problems of silent alpha-thalassemiavs normal, non-deletional alpha-thalassemia vs normal, and deletional alpha-thalassemia vs normal, the best performance of the modelsachieved average ROCAUCs of 0.9505, 0.9430, and 0.9976 in the cross-validation,respectively. In the multiclass model, the best performance achievedan average ROCAUC of 0.9290 in cross-validation. The performance ofour MRM assay and models demonstrated that the hemoglobin subunit mu would play a vital role in screening silent alpha-thalassemiain clinical practice.