Mass Spectrometric Analysis of Urine from COVID-19 Patients for Detection of SARS-CoV-2 Viral Antigen and to Study Host Response

Abstract

SARS-CoV-2 infection has become a major public health burden and affects many organs including lungs, kidneys, the liver, and the brain. Although the virus is readily detected and diagnosed using nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (RT-PCR), detection of its presence in body fluids is fraught with difficulties. A number of published studies have failed to detect viral RNA by RT-PCR methods in urine. Although microbial identification in clinical microbiology using mass spectrometry is undertaken after culture, here we undertook a mass spectrometry-based approach that employed an enrichment step to capture and detect SARS-CoV-2 nucleocapsid protein directly from urine of COVID-19 patients without any culture. We detected SARS-CoV-2 nucleocapsid protein-derived peptides from 13 out of 39 urine samples. Further, a subset of COVID-19 positive and COVID-19 negative urine samples validated by mass spectrometry were used for the quantitative proteomics analysis. Proteins with increased abundance in urine of SARS-CoV-2 positive individuals were enriched in the acute phase response, regulation of complement system, and immune response. Notably, a number of renal proteins such as podocin (NPHS2), an amino acid transporter (SLC36A2), and sodium/glucose cotransporter 5 (SLC5A10), which are intimately involved in normal kidney function, were decreased in the urine of COVID-19 patients. Overall, the detection of viral antigens in urine using mass spectrometry and alterations of the urinary proteome could provide insights into understanding the pathogenesis of COVID-19.

Keywords: COVID-19; SARS-CoV-2; coronavirus; mass spectrometry; quantitative proteomic analysis; urine.

Figure 1.

Experimental workflow for the detection of SARS-CoV-2 using mass spectrometry in urine samples. SARS-CoV-2 positive urine (n = 39) and negative (control) urine (n = 11) samples were centrifuged, inactivated, and subjected to immunoprecipitation (IP) of nucleocapsid protein using a biotinylated antinucleocapsid monoclonal antibody. Rapid trypsin digestion was carried out on IP eluate followed by C₁₈ clean up and LC-MS/MS analysis.

Figure 2.

Summary of the LC-MS/MS analysis for the detection of peptides derived from SARS-CoV-2 nucleocapsid protein. Peptides identified in the urine samples are highlighted in red, while unfilled boxes represent peptides not detected in the indicated samples. Peptides derived from the nucleocapsid protein were not detected in any of the negative control urine samples.

Figure 3.

(A) Peptides identified in LC-MS/MS analysis of SARS-CoV-2 positive urine samples mapped onto nucleocapsid protein domains. RBD = RNA binding domain; SR = serine-arginine-rich; NLS = nuclear localization signal; IDR = intrinsically disordered region. Red block indicates the region of the respective regions of nucleocapsid protein identified by peptides in the LC-MS/MS analysis. Representative MS/MS spectra of the peptides (B) ADETQALPQR, (C) NPANNAAIVLQLPQGTTLPK, (D) QQTVTLLPAADLDDFSK, and (E) KKADETQALPQR identified from the LC-MS/MS analysis of SARS-CoV-2 positive urine samples.

Figure 4.

Quantitative analysis of urinary proteins from SARS-CoV-2 positive and negative individuals. (A) Volcano plot depicting the differentially abundant proteins in urine of SARS-CoV-2 positive individuals compared to negative controls. Statistical analysis was performed by two-sample t test, and proteins were filtered 1.5-fold and for p values < 0.05. (B) Unsupervised clustering of the differentially abundant proteins identified distinct clusters of protein abundance in SARS-CoV-2 positive and negative control individuals.

Figure 5.

Functional characterization of the differentially abundant proteins. Gene ontology enrichment analysis of the differentially abundant proteins was performed by Fisher’s exact test using the DAVID resource. (A) Enriched biological processes among the differentially abundant proteins. (B) Protein–protein interaction network highlighting the enrichment of proteins involved in immune responses among the overabundant proteins in the urine of SARS-CoV-2 positive individuals. Nodes with yellow color are involved in immune response. Nodes with red color belong to the complement system, and nodes with green color belong to the acute phase response. (C) Enriched molecular function terms among the differentially abundant proteins. (D) S-plot representation of the kidney tissue enriched proteins with decreased abundance in the urine of SARS-CoV-2 positive individuals.