A mass spectrometry-based targeted assay for detection of SARS-CoV-2 antigen from clinical specimens
Santosh Renuse,
Patrick M. Vanderboom,
Anthony D. Maus,
Jennifer V. Kemp,
Kari M. Gurtner,
Anil K. Madugundu,
Sandip Chavan,
Jane A. Peterson,
Benjamin J. Madden,
Kiran K. Mangalaparthi,
Dong-Gi Mun,
Smrita Singh,
Benjamin R. Kipp,
Surendra Dasari,
Ravinder J. Singh,
Stefan K. Grebe,
Akhilesh Pandey
Affiliations
Santosh Renuse
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA; Center for Individualized Medicine, Mayo Clinic, Rochester, MN 55905, USA
Patrick M. Vanderboom
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA
Anthony D. Maus
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA
Jennifer V. Kemp
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA
Kari M. Gurtner
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA
Anil K. Madugundu
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA; Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka 560066, India; Manipal Academy of Higher Education, Manipal, Karnataka 576104, India; Center for Molecular Medicine, National Institute of Mental Health and Neurosciences, Hosur Road, Bangalore, Karnataka 560029, India
Sandip Chavan
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA
Jane A. Peterson
Proteomics Core, Medical Genome Facility, Mayo Clinic, Rochester, MN 55905, USA
Benjamin J. Madden
Proteomics Core, Medical Genome Facility, Mayo Clinic, Rochester, MN 55905, USA
Kiran K. Mangalaparthi
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA; Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, Kerala 690525, India
Dong-Gi Mun
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA
Smrita Singh
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA; Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka 560066, India; Manipal Academy of Higher Education, Manipal, Karnataka 576104, India; Center for Molecular Medicine, National Institute of Mental Health and Neurosciences, Hosur Road, Bangalore, Karnataka 560029, India
Benjamin R. Kipp
Department of Laboratory Medicine and Pathology, Division of Laboratory Genetics and Genomics, Mayo Clinic, Rochester, MN 55905, USA
Surendra Dasari
Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA
Ravinder J. Singh
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA; Corresponding authors at: Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, 200 First ST SW, Rochester, MN 55905, USA.
Stefan K. Grebe
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA; Department of Medicine, Division of Endocrinology, Mayo Clinic, Rochester, MN 55902, USA; Corresponding authors at: Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, 200 First ST SW, Rochester, MN 55905, USA.
Akhilesh Pandey
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA; Center for Individualized Medicine, Mayo Clinic, Rochester, MN 55905, USA; Center for Molecular Medicine, National Institute of Mental Health and Neurosciences, Hosur Road, Bangalore, Karnataka 560029, India; Corresponding authors at: Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, 200 First ST SW, Rochester, MN 55905, USA.
Background: The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. Methods: Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. Findings: The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922–0.997) (86/88) sensitivity and 100% (95% CI = 0.958–1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. Interpretation: Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures.
