An LC–HRMS Method for Separation and Identification of Hemoglobin Variant Subunits
Researchers from Stanford University’s School of Medicine and Stanford Health Care report the development of a liquid chromatography high-resolution mass spectrometry (LC–HRMS) method for identifying hemoglobin (Hb) variants. The method can effectively separate several pairs of normal and variant Hb subunits with mass shifts of less than 1 Da and accurately identify them in intact-protein and top-down analyses.
A joint study between the School of Medicine of Stanford University and Stanford Health Care (both located in Stanford, California) has yielded a liquid chromatography high-resolution mass spectrometry (LC–HRMS) method for the separation of several pairs of normal and variant hemoglobin (Hb) subunits with mass shifts of less than 1 Da and has successfully identified them in intact-protein and top-down analyses. As their technique is built on a commercial LC–HRMS system, the researchers state that the process saves on instrument setup time and enables random-access clinical testing. A paper based on this work was published in the Journal of Mass Spectrometry and Advances in the Clinical Lab (1).
A primary respiratory protein in red blood cells which enables the transport of oxygen throughout the body (1), approximately 1400 Hb variants have been identified; some of these can cause symptoms and diseases (2). The identification of Hb variants is (and particularly the variant forms of subunits α and β) therefore of great importance in the clinical diagnosis of hemoglobinopathy (a group of inherited blood disorders involving Hb [3]).
A frequent issue with established approaches for Hb variant identification (including capillary electrophoresis [CE], gel electrophoresis, and high performance liquid chromatography [HPLC]) is their inadequacy regarding the delivery of structural breakdowns of the variants (4). Multiple reports of HRMS methods that improve on traditional methods have been published (5–7); however, when the mass difference between the normal and variant subunits is small, particularly less than 1 Da, ambiguities may occur in analysis, which can lead to failure in Hb variant identification (8).
Utilizing their LC–HRMS method, the researchers achieved LC separation of Hb subunits under denaturing conditions (9) and identified Hb variants following the previously established workflow (8); they specifically credit the use of a C4 reversed-phase column to facilitate the process (1). The LC–HRMS method was also applied to resolve other Hb variant subunits that exhibited more significant mass shifts from their corresponding normal subunits. As expected, it performed successfully and the Hb subunits could be separated in both chromatography and HRMS m/z measurement (1).
The authors state that, with further evaluation to prove the clinical utility, the HRMS method holds the potential to work with or even partially replace conventional methods of Hb variant identification in clinical laboratories (1).
Red blood cells circulating in the blood vessels. © Design Cells - stock.adobe.com
References
1. Chen, A.; Aquino, R. M.; Vidal, H. A.; Wong, C. V.; Luo, R. Y. A Liquid Chromatography-High-Resolution Mass Spectrometry Method for Separation and Identification of Hemoglobin Variant Subunits with Mass Shifts Less Than 1 Da. J. Mass Spectrom. Adv. Clin. Lab. 2025, 35, 1–7. DOI: 10.1016/j.jmsacl.2025.01.002
2. Giardine, B.; Borg, J.; Viennas, E.; et al. Updates of the HbVar Database of Human Hemoglobin Variants and Thalassemia Mutations. Nucleic Acids Res. 2014, 42 (D1), D1063–D1069. DOI: 10.1093/nar/gkt911
3. Hemoglobinopathy. Wikipedia page.https://en.wikipedia.org/wiki/Hemoglobinopathy (accessed 2025-02-28).
4. Xu, M.; Wang, Y.; Xu, A. A Comparative Evaluation of Capillary Electrophoresis, Cation-Exchange High-Performance Liquid Chromatography, and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for the Screening of Hemoglobin Variants. Am. J. Clin. Path. 2021, 156 (3), 445–454. DOI: 10.1093/ajcp/aqaa260
5. Coelho Graça, D.; Hartmer, R.; Jabs, W.; et al. Identification of Hemoglobin Variants by Top-Down Mass Spectrometry Using Selected Diagnostic Product ions. Anal. Bioanal. Chem. 2015, 407, 2837–2845. DOI: 10.1007/s00216-015-8525-5
6. He, L.; Rockwood, A. L.; Agarwal, A. M.; et al. Diagnosis of Hemoglobinopathy and β-thalassemia by 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry of Hemoglobin from Blood. Clin. Chem. 2019, 65 (8), 986–994. DOI:10.1373/clinchem.2018.295766
7. Zhang, J.; Malmirchegini, G. R.; Clubb, R. T.; Loo, J. A. Native Top-Down Mass Spectrometry for the Structural Characterization of Human Hemoglobin. Eur. J. Mass Spectrom. 2015, 21 (3), 221–231. DOI: 10.1255/ejms.1340
8. Luo, R. Y.; Wong, C.; Xia, J. Q.; et al. Neutral-Coating Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Top-Down Identification of Hemoglobin Variants. Clin. Chem. 2023, 69 (1), 56–67. DOI: 10.1093/clinchem/hvac171
9. Donnelly, D. P.; Rawlins, C. M.; DeHart, C. J.; et al. Best Practices and Benchmarks for Intact Protein Analysis for Top-Down Mass Spectrometry. Nat. Methods 2019, 16 (7), 587–594. DOI: 10.1038/s41592-019-0457-0
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