An LC-Immuno-MRM-MS Winterplex Method Development Framework for Respiratory Viral Screening.

Reliable large-scale testing for respiratory viruses, including influenza viruses, coronaviruses, such as SARS-CoV-2, and respiratory syncytial virus (RSV) is essential for both endemic surveillance and pandemic response. While RT-qPCR remains the current gold standard, the COVID-19 pandemic highlighted the need for alternative, scalable detection platforms. Although alternative approaches like liquid chromatography coupled with mass spectrometry (LC-MS) are well-suited for viral multiplexing, they have yet to undergo clinical validation. Here, we present a quantitative immuno-multiple reaction monitoring (iMRM) assay, called Winterplex, for the simultaneous detection of influenza A, influenza B, RSV, and SARS-CoV-2, each targeted via two proteotypic peptides per virus. The method was validated according to ISO standards for in vitro diagnostics and benchmarked against RT-qPCR. To enhance future pandemic preparedness, urgent investment is needed to translate and expand multiplexed MS-based diagnostic methodologies, which offer the flexibility to incorporate a wide range of targets, making them ideally suited for rapid and adaptable responses to emerging viral threats.