Simultaneous detection of respiratory infectious diseases using immunoprecipitation and liquid chromatography-tandem mass spectrometry

ABSTRACT

Background-aim: With recent emergences in new infectious diseases and their variants, there is a need to develop a faster and more specific analytical tool to detect different respiratory infectious diseases such as SARS-CoV-2 or influenza viruses. Not only their symptoms are similar at early stages, but also, they are both enveloped viruses with several common biological properties, often leading to challenges in disease identification. Among different viral components, nucleocapsid protein or nucleoprotein (NP) is highly conserved, less post-translational modifications possessed, and mostly specific for each infectious disease virus types. Therefore, targeting NP could be more advantageous to the method development, achieving much simpler and robust method with minimal subsequent modifications. This study describes a targeted approach for simultaneous detection of NPs from different respiratory infectious diseases using immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple viruses, SARS-CoV-2, influenza virus A and B types, respiratory syncytial virus, and human coronavirus (HCoV-229E), were selected to show that this method can distinguish different disease viruses. Methods: Sample collected via nasopharyngeal swabs in viral transport media was directly subjected to IP using Thermo Scientific™ Pierce™ MS-Compatible IP Kit (Streptavidin). The IP purified samples were then digested using SMART Digest™ Trypsin Kits and analyzed by Thermo Scientific™ Vanquish™ MD HPLC system hyphenated to Thermo Scientific™ TSQ Altis MD mass spectrometer. Data processing was performed using TraceFinder™ LDT software 1.0. Results: Combining IP and LC-MS/MS resulted in a highly targeted approach with the high sensitivity and specificity. The method detected sub tens to hundreds amol of peptides on LC column. Also, it simplified the overall sample preparation process eliminating prior protein precipitation and post sample clean-up. Since the NPs mostly remain unchanged or less modified regardless of variants, the method doesn’t need tremendous alterations once established. Conclusions: This targeted approach can be applied to other enveloped viruses’ detection. Automated IP method is available with KingFisher system so it could lead to a faster turn-around time and higher throughput of the method.