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

Importance of the Topic

Rapid and accurate identification of respiratory infections is critical for patient care, outbreak control and public health surveillance. Traditional assays often require separate tests for each pathogen, leading to extended turnaround times. A multiplexed mass spectrometry approach can streamline diagnostics by detecting multiple viral agents in a single workflow.

Objectives and Study Overview

The study aimed to develop and validate an immunoprecipitation-based, selected reaction monitoring (SRM) LC-MS/MS assay targeting nucleoprotein peptides from SARS-CoV-2, influenza A and B, respiratory syncytial virus (RSV) and HCoV-229E. Key goals included reducing sample preparation to under one hour, shortening chromatographic analysis to five minutes and achieving sub-femtomole sensitivity.

Methodology

Patient swab specimens in viral transport medium were incubated with a panel of biotinylated antibodies specific to each viral nucleoprotein. Antibody–antigen complexes were captured on streptavidin magnetic beads, washed and subjected to on-bead tryptic digestion. Stable-isotope-labeled (SIL) peptide standards were spiked across a calibration range (0.005–10 fmol/μL) to generate quantitation curves. Chromatographic separation employed a rapid gradient from 2 % to 95 % organic mobile phase over 5 minutes. SRM transitions were optimized for two to three peptides per virus, and data processing used strict criteria: accuracy 80–120 %, RSD and CV < 15 %, R2 > 0.99.

Used Instrumentation

• Vanquish MD HPLC system with Hypersil GOLD C18 column (2.1×50 mm, 1.9 μm)
• TSQ Altis MD triple quadrupole mass spectrometer with heated electrospray ionization
• Pierce MS-Compatible IP Kit (Streptavidin) for immunoprecipitation
• SMART Digest Trypsin Kits for on-bead digestion
• TraceFinder LDT software 1.0 for SRM data analysis

Main Results and Discussion

The assay successfully monitored 12 peptides (2–3 per virus) with limits of quantitation between 0.05 and 1 fmol on column and linear ranges extending to 100 fmol. Sample preparation time was reduced from six hours to under one hour, and chromatographic run time to five minutes. Immunoprecipitation provided a clean sample matrix, bypassing conventional protein precipitation and cleanup steps while maintaining high sensitivity and reproducibility.

Benefits and Practical Applications

• High throughput: handles multiple pathogens in a single assay
• Fast turnaround: <1 h prep and 5 min analysis accelerate clinical decision-making
• Robust quantitation: sub-femtomole sensitivity with stringent QC metrics
• Scalable automation: integration with KingFisher systems reduces manual steps by ~70 %

Future Trends and Applications

Ongoing developments may extend this platform to additional respiratory or emerging pathogens, integrate fully automated sample processing, and incorporate machine-learning algorithms for data interpretation. Expanded peptide panels, higher multiplexing capacity and real-time reporting could further enhance clinical diagnostics and pandemic preparedness.

Conclusion

This optimized IP-SRM LC-MS/MS workflow delivers a rapid, sensitive and multiplexed assay for key respiratory viruses. Its streamlined sample preparation, short run time and high analytical performance make it well suited for high-volume clinical and surveillance laboratories.

Reference

• Thermo Fisher Scientific Technical Note 000749