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.

Virulence-associated protein B and C toxin-antitoxin (TA) systems are widespread in prokaryotes, but their precise role in physiology is poorly understood. We have functionally characterized the VapBC22 TA system from Mycobacterium tuberculosis. Transcriptome analysis revealed that overexpression of VapC22 toxin in M. tuberculosis results in reduced levels of metabolic enzymes and increased levels of ribosomal proteins. Proteomics studies showed reduced expression of virulence-associated proteins and increased levels of cognate antitoxin, VapB22 in the ΔvapC22 mutant strain. Furthermore, both the ΔvapC22 mutant and VapB22 overexpression strains of M. tuberculosis were susceptible to killing upon exposure to oxidative stress and showed attenuated growth in guinea pigs and mice. Host transcriptome analysis suggests upregulation of the transcripts involved in innate immune responses and tissue remodeling in mice infected with the ΔvapC22 mutant strain. Together, we demonstrate that the VapBC22 TA system belongs to a key regulatory network and is essential for M. tuberculosis pathogenesis.

Objective To compare COVID-19 acute kidney injury (AKI) to sepsis-AKI (S-AKI) the morphology, transcriptomic and proteomic characteristics of autopsy kidneys were analyzed. Patients and methods Individuals 18 years and older who died from COVID-19 and had an autopsy performed at Mayo Clinic between April 2020 to October 2020 were included. Morphological evaluation of the kidneys of 17 individuals with COVID-19 was performed. In a subset of 7 COVID-19 cases with post-mortem interval of ≤20 hours, ultrastructural and molecular characteristics (targeted transcriptome & proteomics analyses of tubulointerstitium) were evaluated. Molecular characteristics were compared to archived cases of S-AKI and non-sepsis causes of AKI (NS-AKI). Results The spectrum of COVID-19 renal pathology included macrophage dominant microvascular inflammation (glomerulitis and peritubular capillaritis), vascular dysfunction (peritubular capillary congestion & endothelial injury), tubular injury with ultrastructural evidence of mitochondrial damage. Investigation of the spatial architecture using a novel imaging mass cytometry revealed enrichment of CD3 + CD4 + T cells in close proximity to antigen-presenting cells, and macrophage-enriched glomerular and interstitial infiltrates, suggesting an innate and adaptive immune tissue response. COVID-19 AKI and S-AKI, as compared to NS-AKI, had an enrichment of transcriptional pathways involved in inflammation (apoptosis, autophagy, MHC class I and II, and Th1 differentiation). Proteomic pathway analysis demonstrated that COVID-19 AKI & to a lesser extent S-AKI was enriched in necroptosis and sirtuin signaling pathways, both involved in regulatory response to inflammation. Upregulation of ceramide signaling pathway and downregulation of oxidative phosphorylation in COVID-19 AKI was noted. Conclusions This data highlights the similarities between S-AKI and COVID-19 AKI and suggests that mitochondrial dysfunction may play a pivotal role in COVID-19 AKI. This data may allow the development of novel diagnostic and therapeutic targets.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 infection has become a global health pandemic. COVID-19 severity ranges from asymptomatic infection to severe multi-organ disease. Although the inflammatory response has been implicated in the pathogenesis of COVID-19, the exact nature of dysregulation in signaling pathways has not yet been elucidated underscoring the need for further molecular characterization of SARS-CoV-2 infection in humans. Here, we characterize the host response directly at the point of viral entry through analysis of nasopharyngeal swabs. Multiplexed high resolution mass spectrometry-based proteomic analysis of confirmed COVID-19 cases and negative controls identified 7,582 proteins and revealed significant upregulation of interferon-mediated antiviral signaling in addition to multiple other proteins that are not encoded by interferon-stimulated genes (ISGs) or well-characterized during viral infections. Downregulation of several proteasomal subunits, E3 ubiquitin ligases, and components of protein synthesis machinery was significant upon SARS-CoV-2 infection. Targeted proteomics to measure abundance levels of MX1, ISG15, Stat1, RIG-I and CXCL10, detected proteomic signatures of interferon-mediated anti-viral signaling that differentiated COVID-19 positive from negative cases. Phosphoproteomic analysis revealed increased phosphorylation of several proteins with known antiviral properties as well as several proteins involved in ciliary function (CEP131 and CFAP57) that have not previously been implicated in the context of coronavirus infections. Additionally, decreased phosphorylation levels of AKT and PKC, which have been shown to play varying roles in different viral infections, were observed in infected individuals relative to controls. These data provide novel insights that add depth to our understanding of SARS-CoV-2 infection in the upper airway and establish a proteomic signature for this viral infection.