A Multi-omics Longitudinal Study Reveals Alteration of the Leukocyte Activation Pathway in COVID-19 Patients.

Severe coronavirus disease 2019 (COVID-19) infection may lead to lung injury, multi-organ failure, and eventually death. Cytokine storm due to excess cytokine production has been associated with fatality in severe infections. However, the specific molecular signatures associated with the elevated immune response are yet to be elucidated. We performed a mass-spectrometry-based proteomic and metabolomic analysis of COVID-19 plasma samples collected at two time points. Using Orbitrap Fusion LC-MS/MS-based label-free proteomic analysis, we identified around 10 significant proteins, 32 significant peptides, and 5 metabolites that were dysregulated at the severe time points. Few of these proteins identified by quantitative proteomics were validated using the multiple reaction monitoring (MRM) assay. Integrated pathway analysis using distinct proteomic and metabolomic signatures revealed alterations in complement and coagulation cascade, platelet aggregation, myeloid leukocyte activation pathway, and arginine metabolism. Further, we highlight the role of leukocyte activation and arginine metabolism in COVID-19 pathogenesis and targeting these pathways for COVID-19 therapeutics.

Proteomics and Machine Learning Approaches Reveal a Set of Prognostic Markers for COVID-19 Severity With Drug Repurposing Potential.

The pestilential pathogen SARS-CoV-2 has led to a seemingly ceaseless pandemic of COVID-19. The healthcare sector is under a tremendous burden, thus necessitating the prognosis of COVID-19 severity. This in-depth study of plasma proteome alteration provides insights into the host physiological response towards the infection and also reveals the potential prognostic markers of the disease. Using label-free quantitative proteomics, we performed deep plasma proteome analysis in a cohort of 71 patients (20 COVID-19 negative, 18 COVID-19 non-severe, and 33 severe) to understand the disease dynamics. Of the 1200 proteins detected in the patient plasma, 38 proteins were identified to be differentially expressed between non-severe and severe groups. The altered plasma proteome revealed significant dysregulation in the pathways related to peptidase activity, regulated exocytosis, blood coagulation, complement activation, leukocyte activation involved in immune response, and response to glucocorticoid biological processes in severe cases of SARS-CoV-2 infection. Furthermore, we employed supervised machine learning (ML) approaches using a linear support vector machine model to identify the classifiers of patients with non-severe and severe COVID-19. The model used a selected panel of 20 proteins and classified the samples based on the severity with a classification accuracy of 0.84. Putative biomarkers such as angiotensinogen and SERPING1 and ML-derived classifiers including the apolipoprotein B, SERPINA3, and fibrinogen gamma chain were validated by targeted mass spectrometry-based multiple reaction monitoring (MRM) assays. We also employed an in silico screening approach against the identified target proteins for the therapeutic management of COVID-19. We shortlisted two FDA-approved drugs, namely, selinexor and ponatinib, which showed the potential of being repurposed for COVID-19 therapeutics. Overall, this is the first most comprehensive plasma proteome investigation of COVID-19 patients from the Indian population, and provides a set of potential biomarkers for the disease severity progression and targets for therapeutic interventions.

Multiple Reaction Monitoring-Based Targeted Assays for the Validation of Protein Biomarkers in Brain Tumors.

The emergence of omics technologies over the last decade has helped in advancement of research and our understanding of complex diseases like brain cancers. However, barring genomics, no other omics technology has been able to find utility in clinical settings. The recent advancements in mass spectrometry instrumentation have resulted in proteomics technologies becoming more sensitive and reliable. Targeted proteomics, a relatively new branch of mass spectrometry-based proteomics has shown immense potential in addressing the shortcomings of the standard molecular biology-based techniques like Western blotting and Immunohistochemistry. In this study we demonstrate the utility of Multiple reaction monitoring (MRM), a targeted proteomics approach, in quantifying peptides from proteins like Apolipoprotein A1 (APOA1), Apolipoprotein E (APOE), Prostaglandin H2 D-Isomerase (PTGDS), Vitronectin (VTN) and Complement C3 (C3) in cerebrospinal fluid (CSF) collected from Glioma and Meningioma patients. Additionally, we also report transitions for peptides from proteins - Vimentin (VIM), Cystatin-C (CST3) and Clusterin (CLU) in surgically resected Meningioma tissues; Annexin A1 (ANXA1), Superoxide dismutase (SOD2) and VIM in surgically resected Glioma tissues; and Microtubule associated protein-2 (MAP-2), Splicing factor 3B subunit 2 (SF3B2) and VIM in surgically resected Medulloblastoma tissues. To our knowledge, this is the first study reporting the use of MRM to validate proteins from three types of brain malignancies and two different bio-specimens. Future studies involving a large cohort of samples aimed at accurately detecting and quantifying peptides of proteins with roles in brain malignancies could potentially result in a panel of proteins showing ability to classify and grade tumors. Successful application of these techniques could ultimately offer alternative strategies with increased accuracy, sensitivity and lower turnaround time making them translatable to the clinics.

Proteomic investigation reveals dominant alterations of neutrophil degranulation and mRNA translation pathways in patients with COVID-19.

The altered molecular proteins and pathways in response to COVID-19 infection are still unclear. Here, we performed a comprehensive proteomics-based investigation of nasopharyngeal swab samples from patients with COVID-19 to study the host response by employing simple extraction strategies. Few of the host proteins such as interleukin-6, L-lactate dehydrogenase, C-reactive protein, Ferritin, and aspartate aminotransferase were found to be upregulated only in COVID-19-positive patients using targeted multiple reaction monitoring studies. The most important pathways identified by enrichment analysis were neutrophil degranulation, interleukin-12 signaling pathways, and mRNA translation of proteins thus providing the detailed investigation of host response in COVID-19 infection. Thus, we conclude that mass spectrometry-detected host proteins have a potential for disease severity progression; however, suitable validation strategies should be deployed for the clinical translation. Furthermore, the in silico docking of potential drugs with host proteins involved in the interleukin-12 signaling pathway might aid in COVID-19 therapeutic interventions.