Rapid Simultaneous Detection of Respiratory Infectious Diseases using Immunoprecipitation and Liquid Chromatography-Tandem Mass Spectrometry

Importance of the topic

Rapid detection of respiratory pathogens is vital for clinical diagnostics and public health surveillance.
Traditional methods can be time-consuming and may lack multiplexing capability.
Combining immunoprecipitation with targeted LC-MS/MS can address these limitations by enriching viral proteins and enabling simultaneous analysis of multiple agents in under one hour.

Study objectives and overview

The study aimed to design and validate a fast, sensitive workflow that monitors core nucleoproteins from SARS-CoV-2, influenza A, influenza B, respiratory syncytial virus (RSV), and human coronavirus 229E in nasopharyngeal swab samples.
Key goals included reducing sample preparation time from six hours to under one hour and achieving a five-minute LC-MS run while maintaining high analytical performance.

Methodology

An antibody panel was biotinylated and pooled to immunoprecipitate target nucleoproteins directly from viral transport medium.
On-bead trypsin digestion was optimized to 15 minutes at 70 °C to generate peptide fragments.
Stable isotope-labeled peptides were spiked for calibration and quantitation via selected reaction monitoring (SRM).
Data processing was performed using TraceFinder™ LDT software with criteria of accuracy ± 20 %, RSD < 15 %, and R2 > 0.99.

Used instrumentation

• Thermo Scientific™ Vanquish™ MD HPLC system with Hypersil™ GOLD C18 column (2.1 × 50 mm, 1.9 μm)
• Thermo Scientific™ TSQ Altis™ MD mass spectrometer with heated electrospray ionization
• SMART Digest™ Trypsin Kit and Pierce™ streptavidin magnetic IP kit for sample preparation

Main results and discussion

The workflow successfully monitored 12 signature peptides (2–3 per virus) across a retention window of 1.45–2.48 minutes.
Shortened IP incubation (30 minutes) and digestion (15 minutes) steps still yielded comparable peptide recovery.
Limits of detection ranged from 0.05 to 1 fmol on column, with linear dynamic ranges up to 100 fmol and R² values above 0.99.
Retention time variation was under ± 0.01 minutes, confirming run-to-run stability.

Benefits and practical applications

• High-throughput analysis with total assay time below one hour
• Multiplexed detection reduces reagent usage and labor compared to single-analyte assays
• Clean sample matrix via IP eliminates protein precipitation and extensive clean-up
• Suitable for clinical laboratories and outbreak monitoring

Future trends and potential applications

Integration with automated platforms like KingFisher™ can further reduce manual steps and increase throughput.
The approach could be extended to other enveloped viruses or emerging pathogens by updating antibody panels and peptide targets.
Advancements in MS instrumentation may allow sub-femtomole sensitivity and shorter gradients for point-of-care testing.

Conclusion

This study demonstrates a robust IP-SRM workflow enabling rapid, multiplexed detection of key respiratory viruses with excellent sensitivity and reproducibility.
The streamlined protocol supports urgent diagnostic needs and can be scaled for high-volume laboratories.

References

1. Dutta N.K., Mazumdar K., Gordy J.T.; The Nucleocapsid Protein of SARS–CoV-2: A Target for Vaccine Development. Journal of Virology 2020, 94(13).
2. Heiny A.T., Miotto O., Srinivasan K.N., et al.; Evolutionarily Conserved Protein Sequences of Influenza A Viruses as Vaccine Targets. PLoS ONE 2007, 2(11).
3. Centers for Disease Control and Prevention; Preparation of Viral Transport Medium. CDC 2019.