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.
Background We evaluated the analytical sensitivity and specificity of four rapid antigen diagnostic tests (Ag RDTs) for SARS-CoV-2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method; and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test. Methods 350 (150 negative and 200 RT-qPCR positive) residual phosphate buffered saline (PBS) samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR positive, antigen positive samples for further analysis. Results All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1,500,000 copies/mL RNA, and detected ≥75% of samples with viral load of 500,000 to 1,500,000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50,000-500,000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load <50,000 copies/mL compared to the BD Veritor. Among samples with detectable antigen and viral load <50,000 copies/mL, sensitivity of the Ag RDT varied between 13.0% (BD Veritor) and 78.3% (ACON FIA). Conclusions Ag RDTs differ significantly in analytical sensitivity, particularly at viral load <500,000 copies/mL.
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 diagnostics including RT-PCR-based assays, antigen detection by lateral flow assays and antibody-based assays have been developed and deployed in a short time. However, many of these assays are lacking in sensitivity and/or specificity. Here, we describe an immunoaffinity purification followed by high resolution mass spectrometry-based targeted assay capable of detecting viral antigen in nasopharyngeal swab samples of SARS-CoV-2 infected individuals. 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 ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assays on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was created using fragment ion intensities in the PRM data. This resulted in 97.8% sensitivity and 100% specificity with RT-PCR-based molecular testing as the gold standard. Our results demonstrate that direct detection of infectious agents from clinical samples by mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories.
COVID-19 vaccines are becoming more widely available, but accurate and rapid testing remains a crucial tool for slowing the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. Although the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains the most prevalent testing methodology, numerous tests have been developed that are predicated on detection of the SARS-CoV-2 nucleocapsid protein, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay-based approaches. The continuing emergence of SARS-CoV-2 variants has complicated these approaches, as both qRT-PCR and antigen detection methods can be prone to missing viral variants. In this study, we describe several COVID-19 cases where we were unable to detect the expected peptide targets from clinical nasopharyngeal swabs. Whole genome sequencing revealed that single nucleotide polymorphisms in the gene encoding the viral nucleocapsid protein led to sequence variants that were not monitored in the targeted assay. Minor modifications to the LC-MS/MS method ensured detection of the variants of the target peptide. Additional nucleocapsid variants could be detected by performing the bottom-up proteomic analysis of whole viral genome-sequenced samples. This study demonstrates the importance of considering variants of SARS-CoV-2 in the assay design and highlights the flexibility of mass spectrometry-based approaches to detect variants as they evolve.
Aims The identification and differentiation of antibiotic‐resistant bacteria by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) profiling remains a challenge due to the difficulty in detecting unique protein biomarkers associated with this trait. To expand the detectable proteome in antibiotic‐resistant bacteria, we describe a method implementing offline LC protein separation/fractionation prior to MALDI‐ToF‐MS and top‐down MALDI‐ToF/ToF‐MS (tandem MS or MS/MS) for the analysis of several antibiotic‐resistant Escherichia coli isolates. Methods and Results Coupling offline LC with MALDI‐ToF‐MS increased the number of detected protein signals in the typically analyzed mass regions (m/z 3000–20 000) by a factor of 13. Using the developed LC‐MALDI‐ToF‐MS protocol in conjunction with supervised principal components analysis, we detected a protein biomarker at m/z 9355 which correlated to β‐lactam resistance among the E. coli bacteria tested. Implementing a top‐down MALDI‐ToF/ToF‐MS approach, the prefractionated protein biomarker was inferred as a DNA‐binding HU protein, likely translated from the blaCMY‐2 gene (encoding AmpC‐type β‐lactamase) in the incompatibility plasmid complex A/C (IncA/C). Conclusions Our results demonstrate the utility of LC‐MALDI‐MS and MS/MS to extend the number of proteins detected and perform MALDI‐accessible protein biomarker discovery in microorganisms. Significance and Impact of the Study This outcome is significant since it expands the detectable bacterial proteome via MALDI‐ToF‐MS.
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