Exploring novel biomarkers of COVID-19 diagnosis and aggravation by comprehensive analysis of modified nucleosides, and development of automated analysis system

Importance of the Topic

Analysis of modified nucleosides in biological fluids offers a low‐risk alternative to PCR for COVID-19 detection and provides insight into disease severity. Variations in these chemical markers reflect viral replication and host response, enabling both diagnosis and prognosis assessment in a rapid, minimally invasive manner.

Study Objectives and Overview

The study aimed to identify novel serum and urine biomarkers among modified nucleosides for COVID-19 diagnosis and severity prediction. A secondary goal was to develop and validate a fully automated LC-MS/MS workflow integrating sample pretreatment and analysis for high-throughput clinical application.

Methodology and Instrumentation

• Sample Sources: SARS-CoV-2‐infected human cell cultures, serum and urine from PCR-positive COVID-19 patients (including mild cases) and healthy controls.
• Pretreatment: Protein precipitation, target extraction and dilution were automated using the CLAM-2030 platform, reducing manual processing from ~30 to ~6 minutes and further down to ~5 minutes after optimization.
• Chromatography and Detection: Shimadzu Nexera X2 UHPLC coupled to LCMS-8050. ODS column, gradient elution at 0.3 mL/min, injection volume 2 µL, column temperature 40 °C. ESI positive mode with DL 250 °C, heat block 400 °C, interface 300 °C, nebulizing gas 3 L/min, drying gas 10 L/min.
• Validation: Linearity (0.1–100 nM, R² > 0.999), calibration accuracy within ±25% at LOQ and ±10% at higher concentrations; retention time CV < 0.1% for t6A/ms2t6A and < 1% for adenosine; area reproducibility CV < 5%; serum recovery within ±25% (CV < 6%).

Main Results and Discussion

• Diagnostic Biomarkers: Two modified adenosines, N⁶-threonylcarbamoyladenosine (t6A) and 2-methylthio-N⁶-threonylcarbamoyladenosine (ms2t6A), were significantly elevated in COVID-19 patients compared to healthy controls, with sensitivity and specificity comparable to PCR.
• Prognostic Indicator: Elevated serum ms2t6A levels at admission correlated with subsequent clinical deterioration even among initially mild cases, suggesting early prediction of aggravation.
• Automated Performance: The CLAM-2030–based workflow achieved total turnaround of ~10 minutes (pretreatment plus analysis), reproducing manual assay results with high precision and accuracy.

Benefits and Practical Applications

• Rapid, high‐throughput screening for COVID-19 infection and severity risk.
• Reduced biosafety hazards by analyzing serum or urine rather than respiratory specimens.
• Automated operation minimizes labor, standardizes results, and supports clinical decision making for patient triage and treatment planning.

Future Trends and Potential Applications

• Expansion to larger biomarker panels encompassing additional modified nucleosides or other metabolites for multi‐disease profiling.
• Integration with point‐of‐care devices and AI‐driven data analysis for on-site decision support.
• Adaptation of the automated platform for monitoring treatment response, vaccine efficacy or emerging viral variants.
• Miniaturization and microfluidic coupling for real-time, continuous monitoring in clinical settings.

Conclusion

The comprehensive analysis identified t6A and ms2t6A as reliable markers for COVID-19 diagnosis and early prediction of disease aggravation. The fully automated LC-MS/MS system achieved rapid, reproducible quantification from pretreatment through analysis, offering a powerful tool for clinical management and epidemic control.

Instrumentation Used

• Shimadzu Nexera X2 UHPLC system
• Shimadzu LCMS-8050 triple quadrupole mass spectrometer
• CLAM-2030 automated sample pretreatment platform