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.