MSACL 2023 Abstract

Introduction:

We are now living in a new era with new normal after the COVID-19 pandemic. Recently, respiratory syncytial virus (RSV) has been another concern as it surges among children. In addition to influenza viruses, not only are their symptoms similar at early stages, but they are also both enveloped viruses with several common biological properties, often leading to challenges in accurate identification. Thus, there is a need to develop a faster and more specific analytical tool that can differentiate infectious diseases.

Among different viral components, nucleocapsid protein or nucleoprotein (NP) is highly conserved and specific for infectious disease virus types. Therefore, targeting NP could provide a more robust and faster way for disease identification.

This study describes a targeted approach for the simultaneous detection of NPs from different respiratory infectious diseases using immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Objectives:

To monitor multiple infectious diseases in a fast and sensitive way using immunoprecipitation and selected reaction monitoring.

Methods:

Prior to IP, equal amounts of all biotinylated antibodies were pooled together as one antibody panel for this study. The biotinylated antibody panel was added to samples collected via nasopharyngeal swabs in viral transport media (VTM) followed by incubation for 15 minutes at 25 C degree with rotation. The antigen-antibody complex in VTM was directly subjected to IP using Thermo Scientific™ Pierce™ MS-Compatible IP Kit (Streptavidin).

The IP purified samples were then digested for 15 minutes at 70 C degree with vortexing at 1000 rpm using SMART Digest™ Trypsin Kits and analyzed by Thermo Scientific™ Vanquish™ MD HPLC system hyphenated to Thermo Scientific™ TSQ Altis MD mass spectrometer. Data acquisition, processing and reporting were performed using TraceFinder™ LDT software 1.0.

Results:

Multiple viruses, SARS-CoV-2, influenza virus A and B types, RSV, and human coronavirus (HCoV-229E), were selected to show that this method can distinguish different disease viruses. The protein sequences of those selected NPs are mostly unique to each disease type and remain unchanged regardless of their variants, resulting in a reliable target peptides list for selected reaction monitoring (SRM). Combining with IP which uses a specific antigen-antibody interaction, it provides a highly targeted and confident detection of each disease. Thus, this approach is much simpler and robust with minimal changes when compared to other methods targeting different components such as the spike protein.

The workflow was optimized from sample preparation to LC-MS analysis. The protein precipitation and post sample clean-up were eliminated. From IP procedure, two incubation steps for antigen-antibody complex formation and immobilization on the magnetic beads were reduced to 15 minutes each (originally 1 hour each). Trypsin digestion incubation time was optimized to 15 minutes (previously 90 minutes). Particularly, the reduction of trypsin digestion time was achieved owing to a generation of much cleaner sample matrix by IP. The entire process was finalized to less than 1 hour from 4 hours. LC-MS run time was also optimized to 5 minutes.

A total of 12 peptides were successfully monitored (2 to 3 peptides per disease) by SRM. Calibration curve was generated with stable isotope-labeled standards (Thermo Scientific HeavyPeptide AQUA Ultimate). With criteria of % RSD < 15, CV < 15, and R2 > 0.99, LOQs were determined to be between 0.05 and 1 fmol of peptides on LC column with retention time variation ± 0.01 minutes. The method significantly improved the sensitivity and turn-around time, compared to other digestion or peptide enrichment methods.

Conclusion:

• Simple and robust approach by targeting the nucleoprotein component of the enveloped virus.
• Quick sample preparation taking < 1 hour and virus detection using 5-minute LC-MS method.
• MS-compatible and clean sample matrix generation by immunoprecipitation.
• Highly targeted, sensitive, and confident detection by immunoprecipitation and selected reaction monitoring.

For Research Use Only – Not For Diagnostic Procedures.