Enhancing detection of hemoglobin variants in clinical research using dried blood spot and high-resolution accurate mass (HRAM) Orbitrap mass spectrometry

Importance of Topic

Diseases caused by hemoglobin variants and thalassemia represent some of the most common genetic disorders worldwide. Rapid and accurate identification of these variants is critical for early diagnosis, treatment monitoring and epidemiological studies. Traditional electrophoretic and chromatographic assays often lack the resolution to distinguish closely related hemoglobin subunits or novel mutations. High-resolution accurate-mass top-down mass spectrometry combined with dried blood spot sampling offers a minimally invasive, high-throughput solution that enhances both identification and quantitation of hemoglobin variants.

Objectives and Study Overview

This study aimed to establish a top-down HRAM LC-MS workflow for the detection and quantitation of hemoglobin variants from dried blood spots. Key objectives included:

• Developing a rapid protein extraction and precipitation protocol in a 96-well plate format
• Applying a Thermo Scientific Orbitrap Exploris 240 mass spectrometer with BioPharma option for intact protein analysis
• Leveraging ProSightPD software for variant identification and TraceFinder software for quantitative analysis

Methodology and Instrumentation

A streamlined sample preparation protocol was used:

• Spotting 10 µL of blood calibrator onto filter cards and drying at room temperature
• Punching a 3.2 mm disc into a 96-well plate and extracting hemoglobin with water and acetonitrile precipitation
• Resolubilizing proteins for LC-MS injection

Chromatography and mass spectrometry were performed as follows:

• Vanquish Flex UHPLC system with MAbPac RP column (1 × 100 mm, 4 µm) at 80 °C
• Gradient elution using 0.1% formic acid and 0.02% trifluoroacetic acid
• Orbitrap Exploris 240 operated at 120,000 resolution for top-down data-dependent acquisition
• ProSightPD for sequence mapping and mutation confirmation
• TraceFinder for peak extraction and quantitation, monitoring the most abundant isotopes of charge states +18 and +19

Key Results and Discussion

• All four hemoglobin chains (alpha, beta, variant beta, delta) were detected and resolved, with a 1.6 m/z shift at charge state +19 between normal and Hb S beta chains
• Sequence coverage exceeded 40% for both normal beta and Hb S beta, confirming the β6 Glu→Val substitution; delta chain detection demonstrated sensitivity to low-abundance species
• Calibration curves over 2.5–100 mg/mL exhibited excellent linearity (R² > 0.99), LOD of 1.0 mg/mL and LOQ of 2.5 mg/mL
• Inter-day precision and accuracy were within acceptable limits (RSD < 8%, accuracy 73–112%)

Benefits and Practical Applications

• Minimal sample preparation (< 1 hour) amenable to automation in 96-well format
• High resolving power enables discrimination of isobaric variants and detection of novel mutations
• Single-platform workflow for simultaneous qualitative and quantitative analysis
• Suitable for large-scale clinical studies, newborn screening and longitudinal patient monitoring

Future Trends and Opportunities

• Full integration with automated liquid handling for ultra-high throughput screening
• Multiplexed assays targeting additional protein biomarkers from DBS
• Advanced bioinformatics pipelines for rapid variant annotation and reporting
• Deployment in point-of-care testing and global health surveillance programs

Conclusion

The top-down HRAM LC-MS workflow using dried blood spots and an Orbitrap Exploris 240 platform provides a robust, sensitive and high-throughput solution for the detection and quantitation of hemoglobin variants. This approach overcomes limitations of traditional assays and supports comprehensive analysis of common and rare hemoglobinopathies in clinical research contexts.

References

1. Bain BJ. Haemoglobinopathy Diagnosis. 2nd ed. Blackwell Science; 2006:1–26.
2. Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ. 2008;86:480–487.
3. Billett HH. Hemoglobin and Hematocrit. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd ed. Butterworths; 1990:Chapter 151.