Toxin-like peptides in plasma, urine and faecal samples from COVID-19 patients

Abstract

Background: SARS-CoV-2 that causes COVID-19 disease and led to the pandemic currently affecting the world has been broadly investigated. Different studies have been performed to understand the infection mechanism, and the involved human genes, transcripts and proteins. In parallel, numerous clinical extra-pulmonary manifestations co-occurring with COVID-19 disease have been reported and evidence of their severity and persistence is increasing. Whether these manifestations are linked to other disorders co-occurring with SARS-CoV-2 infection, is under discussion. In this work, we report the identification of toxin-like peptides in COVID-19 patients by application of the Liquid Chromatography Surface-Activated Chemical Ionization - Cloud Ion Mobility Mass Spectrometry. Methods: Plasma, urine and faecal samples from COVID-19 patients and control individuals were analysed to study peptidomic toxins' profiles. Protein precipitation preparation procedure was used for plasma, to remove high molecular weight proteins and efficiently solubilize the peptide fraction; in the case of faeces and urine, direct peptide solubilization was employed. Results: Toxin-like peptides, almost identical to toxic components of venoms from animals, like conotoxins, phospholipases, phosphodiesterases, zinc metal proteinases, and bradykinins, were identified in samples from COVID-19 patients, but not in control samples. Conclusions: The presence of toxin-like peptides could potentially be connected to SARS-CoV-2 infection. Their presence suggests a possible association between COVID-19 disease and the release in the body of (oligo-)peptides almost identical to toxic components of venoms from animals. Their involvement in a large set of heterogeneous extra-pulmonary COVID-19 clinical manifestations, like neurological ones, cannot be excluded. Although the presence of each individual symptom is not selective of the disease, their combination might be related to COVID-19 by the coexistence of the panel of the here detected toxin-like peptides. The presence of these peptides opens new scenarios on the aetiology of the COVID-19 clinical symptoms observed up to now, including neurological manifestations.

Keywords: COVID-19; SARS-CoV-2; toxin-like peptides.

Figure 1.

( a) Base peak LC Full Scan (MS), tandem mass (MS/MS) chromatogram of an extracted plasma sample of a patient and a control subject and ( b) a blow-up of a specific chromatogram region (5.713–5.719 min). The blow-up shows the four regions of data acquisition: 1) Full scan mass spectrum originated by the cloud containing low m/z ratio molecular species; 2) Tandem mass spectra (MS/MS) mass spectrum originated by the cloud containing low m/z ratio molecular species; 3) Full scan mass spectrum originated by the cloud containing medium-high (MedHigh) m/z ratio molecular species; 4) Tandem mass spectra (MS/MS) mass spectrum originated by the cloud containing medium-high (MedHigh) m/z ratio molecular species.

Figure 2.

Examples of full scan mass spectra, obtained by analysing a COVID-19 positive urine sample and acquired focalizing solvent ion cloud species containing a) low, b) high m/z species extracted in the 5.713–5.719 min chromatographic region and ESI full scan mass spectrum obtained analysing the same sample and extracting the signal at the same retention time extracting c) low and d) high m/z ratio.

Figure 3.. Examples of mass spectra peptide characterization together with the peptide ion fragmentation pathways.

Example of how MS/MS signal were assigned to the different N-terminal y,z (blue and purple colour in panel a) and c-terminal b,c (red and yellow colour) fragmentation series (detailed in panel b). Only mass spectra exhibiting a statistical -log(e) score higher that 10 and a false discovery rate lower than 0.05 were considered for the identification (reported in panel c). False discovery rate and statistical score were estimated by means of reverse sequence approach.

Figure 4.. Alignment of toxin-like peptides to Conotoxin Pu6.1 precursor.

Conotoxin Pu6.1 precursor from Conus pulicarius (UniprotKB:D2DGD8) is aligned with the toxin-like peptides identified in four out of five plasma samples. Being the protein secreted and cleaved, leader-region pro-peptide and mature cysteine rich domains are highlighted in green, yellow and red, respectively. The shown peptides correspond to the longest observed peptides, as we did not make any specific selection for secreted proteins, precursors are expected to be present in our samples. Each identified toxin-like peptide is named according to the sample of origin and its uniqueness. For each of them, the number reported in square brackets indicates the number of identical toxin-like peptides identified in the same sample.