Untargeted Mass Spectrometry Approach to Study SARS-CoV-2 Proteins in Human Plasma and Saliva Proteome

Abstract

Since the start of the COVID-19 outbreak, more than four million people have died of this disease. Given its ability to provide a precise response, mass spectrometry-based proteomics could represent a useful tool to study this pathology. To this end, an untargeted nLC-ESI-MS/MS-based method to characterise SARS-CoV-2 proteins, including possible variants, and investigate human saliva and plasma proteome in a single analysis was developed for further application in patients. Four SARS-CoV-2 recombinant proteins, three (S1–S2–RBD) belonging to the spike glycoprotein (S) and one corresponding to the nucleoprotein (N), were prepared and analysed with nLC-UHRTOF by injecting decreasing amounts to establish the limit of detection (LOD) of the method. This was determined as 10 pg for all the components of the S protein and for N (71 amol and 213 amol, respectively). Various viral inactivation strategies plus deglycosylation and digestion approaches were then tested in saliva and plasma spiked with different quantities of SARS-CoV-2 recombinant proteins. The limit of characterisation (LOC) in saliva for the N and S proteins was observed at 100 pg (coverage of 20% and 3%, respectively); instead, in plasma, it was 33 pg for N and 330 pg for the S protein, with a coverage of 4% for both. About 300 and 800 human proteins were identified in plasma and saliva, respectively, including several key effectors and pathways that are known to be altered in COVID-19 patients. In conclusion, this approach allows SARS-CoV-2 proteins and the human proteome to be simultaneously explored, both for plasma and saliva, showing a high relevant potential for retrospective studies aimed at investigating possible virus variants and for patient stratification.

Proteomic snapshot of saliva samples predicts new pathways implicated in SARS-CoV-2 pathogenesis

“…To the best of our knowledge, only one study focused on the application of untargeted mass spectrometry to compare the saliva proteome of SARS-CoV-2 infected and Non-COVID individuals. Although differences in the expression of a human protein related to the defense/immunity protein, an immunoglobulin, a translational protein, and a protease inhibitor were observed, no clear link with the mechanisms of pathogenesis and response to SARS-CoV-2 could be established [32]. This is an exploratory study aimed to describe DE proteins that could be related to COVID-19 disease susceptibility.…”

Proteomic snapshot of saliva samples predicts new pathways implicated in SARS-CoV-2 pathogenesis

“…To the best of our knowledge, only one study has focused on the application of untargeted mass spectrometry to compare the saliva proteome of SARS-CoV-2-infected and non-COVID-19 individuals. Although differences in the expression of a human protein related to the defense/immunity protein, an immunoglobulin, a translational protein, and a protease inhibitor were observed, no clear link with the mechanisms of pathogenesis and response to SARS-CoV-2 could be established [ 32 ].…”

“…However, we did not see these differences in the main comparison of groups: NcNs vs NcSus. Furthermore, as a quality control of our study, to determine the identified human proteins with the highest confidence we filtered and selected only the proteins appearing in two experiments with two internal standards, and even doing that, we obtained similar or even higher numbers compared to previous studies in nasopharyngeal swabs [ 11 ] and in saliva samples [ 32 ]). It also constitutes one of the best studies reporting the combined human proteome and microbial meta-proteome in saliva samples, since no previous study reported alterations in canonical human and microbial pathway analysis in saliva samples.…”

Plasma Proteomic Variables Related to COVID-19 Severity: An Untargeted nLC-MS/MS Investigation

“…To allow safe sample manipulation, the virus was inactivated and plasma samples were digested by following the protocol described by Pagani et al [ 58 ]. Briefly, the inactivation was performed by heating samples for 60 min at 56 °C and by adding freezing ethanol (Honeywell, ≥99.8%, Offenbach, Germany), 9:1 ethanol/sample ( v / v ).…”

“…The eluted peptides were ionised using a nanoBoosterCaptiveSpray™ (Bruker Daltonics) source using heated nitrogen dry gas (T = 150 ℃; 3 L/min) enriched with acetonitrile (ACN) (Honeywell, ≥99.9%, Offenbach, Germany). The mass spectrometer was operated in DDA (Data Dependent Acquisition Mode), with automatic switching between full-scan MS and MS/MS acquisition, as already reported [ 58 ]. N2 was used as a gas for CID (collision-induced dissociation) fragmentation.…”