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

Lisa Pagani,Allia Mahajneh,Andrea Lombardi,Paola Gruarin,Fulvio Magni,Lucrezia Criscuolo,Clizia Chinello,Francesco Blasi,Francesca Clerici,Antonio Muscatello,Riccardo Ungaro,Andrea Gori,Alessandra Bandera,Renata Grifantini,Andrea Favalli

doi:10.3390/biochem2010005

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.

Highlights

SARS-CoV-2 is the etiologic agent responsible for coronavirus disease 2019 (COVID-19).About 80% of patients infected by SARS-CoV-2 are asymptomatic or present mild symptoms, but they can infect other people
We investigated the ability of a method based on nLC-ESI-mass spectrometry (MS)/MS to simultaneously identify specific SARS-CoV-2 proteins and characterise the host human proteome both in saliva and plasma samples with the perspective of a future application in detecting possible variants of this virus and in clustering COVID-19 patients with different outcomes in a single analysis
The experimental workflow was finalised to obtain a protocol able to simultaneously detect and characterise SARS-CoV-2 proteins and investigate the alterations of human saliva and plasma proteome of the patients affected by COVID-19

Summary

Introduction

SARS-CoV-2 is the etiologic agent responsible for coronavirus disease 2019 (COVID-19). About 80% of patients infected by SARS-CoV-2 are asymptomatic or present mild symptoms, but they can infect other people. Some infected individuals which are symptomatic may present more severe symptoms which could lead to death [1]. Symptoms appear 2–14 days after the infection. Since the virus can affect both lungs, patients present signs and symptoms which are associated with viral pneumonia BioChem 2022, 2 fever, cough, sore throat, headache, fatigue, myalgia and dyspnoea). Loss of smell or taste and gastrointestinal disorders have been reported in some cases [1,2]

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