Establishment of a Top-Down Liquid Chromatography-High-Resolution-Mass Spectrometry Method With Multiple...

Identification of hemoglobin (Hb) variants is of significant value in the clinical diagnosis of hemoglobinopathies. A common issue with the conventional methods for identifying Hb variants is their limited resolution to provide primary structures. Previous literature has shown the ability for high-resolution mass spectrometry (HR-MS) methods to accurately identify Hb variants, but ambiguity may still remain when distinguishing isomeric Hb variants due to inadequate AA sequence coverage. Using multiple fragmentation modes can be a solution to achieve higher sequence coverage in MS² fragment analysis. A liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) method with multiple fragmentation modes was developed for identifying Hb variants, which could both effectively separate pairs of normal and variant Hb subunits and accurately distinguish isomeric Hb variant subunits. The separation was facilitated by a C4 reversed-phase column, and the fragmentation modes in use were higher-energy collision dissociation (HCD) and electron-transfer/higher-energy collision dissociation (EThcD). The fragmentation modes HCD, ETD, and EThcD were tested with normal α and β subunits to evaluate the MS² performance of each mode. The combined use of EThcD and HCD was able to make high AA sequence coverage. Five clinical samples, including heterozygous Hb E and Hb Constant Spring (CS), Hb Hofu, Hb Raleigh, Hb P-Nilotic, and Hb F-Kuala Lumpur (KL), were analyzed to demonstrate the performance of the LC-HR-MS method for accurately identifying Hb variants samples. This article demonstrated the advantages of LC-HR-MS with multiple fragmentation modes in effectively solving clinical cases of Hb variants. In clinical practice, the combination of the LC-HR-MS and conventional Hb test methods is favorable, allowing for accurate diagnosis of Hb variants and significantly reducing the need for more costly genetic tests.