Enhanced Detection of Hemoglobin Variants in Clinical Research by DBS and High-Resolution Accurate Mass (HRAM) MS

Significance of the Topic

The detection of hemoglobin variants is essential for the accurate diagnosis and monitoring of common genetic blood disorders such as sickle cell disease and thalassemias. Traditional assays often lack the resolution needed to distinguish closely related proteoforms and isobaric variants. Integrating dried blood spot sampling with high-resolution accurate-mass top-down mass spectrometry offers a streamlined workflow for high throughput clinical research.

Objectives and Study Overview

This study aimed to establish a top-down mass spectrometry approach using dried blood spots and the Orbitrap Exploris 240 system for enhanced identification and quantitation of hemoglobin variants. Key goals included minimizing sample preparation time, automating the workflow in 96-well format, and demonstrating confident variant sequencing and ratio-based quantitation.

Methodology and Instrumentation

A 3.2 mm disc was punched from EDTA-stabilized whole blood spots and subjected to direct protein extraction and precipitation in a 96-well plate. Separation was achieved on a Vanquish Flex UHPLC with a MAbPac RP column under a stepped gradient of aqueous formic acid/TFA and organic phase. Intact proteins were analyzed on an Orbitrap Exploris 240 with BioPharma option. Sequence identification employed ProSightPD with a customized database, and quantitative analysis of isotopic precursors was performed using TraceFinder 5.1.

Main Results and Discussion

The top-down workflow successfully resolved and sequenced alpha, beta, gamma, delta, and variant chains, including those differing by less than 1 Da. Sequence coverage exceeded 90 % for major chains. Quantitation of hemoglobin S spiked into blood achieved linear calibration over 2.5–100 mg/mL with R2 values above 0.994 and precision below 8 %. Daily limits of detection and quantitation were consistent at 1.0 and 2.5 mg/mL, respectively.

Benefits and Practical Applications

• Minimal sample preparation (< 1 hr) compatible with automated robotics
• High‐confidence variant characterization via intact top-down sequencing
• Accurate ratio-based quantitation of target chains
• Scalable 96-well format for clinical research throughput

Future Trends and Opportunities

Advances may include integration with fully automated sample handling systems, expansion of variant databases for novel proteoforms, and coupling top-down workflows with targeted MS/MS for deeper structural insights. Translation to diagnostic laboratories could further improve newborn screening and patient stratification.

Conclusion

The described top-down LC-HRAM MS method enables rapid, automated, and high-resolution detection and quantitation of hemoglobin variants directly from dried blood spots, offering a robust platform for clinical research applications.

References

• Song YE, Hassell K, Goucher E, Guo J, Correa T. Enhanced Detection of Hemoglobin Variants in Clinical Research by DBS and High-Resolution Accurate-Mass MS. Thermo Fisher Scientific; 2024.