Identification of SARS-CoV-2 from Human Lung Formalin-Fixed Paraffin-Embedded Tissue Sections Using Mass Spectrometry

The COVID-19 pandemic affected hundreds of million people worldwide with a significant mortality rate. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), enters the epithelial cells lining the upper respiratory tract by binding to the ACE2 receptor on host cells through the receptor binding domain of its spike protein. This process is facilitated by host protein transmembrane serine protease 2 (TMPRSS2) and various lysosomal proteases. Viral infection of host cells is followed by the production of pro-inflammatory cytokines and immune cell recruitment as part of the acute inflammatory response.

Although a limited number of studies have reported direct identification of virus from human tissues based on viral RNA (Majumdar et al., 2022), viral proteins by immunohistochemistry (IHC) (Roden et al., 2021), or electron microscopy (Bullock et al., 2022), direct evidence of viral proteins from host tissues using mass spectrometry has not been reported thus far. In this study, using mass spectrometry, we sought to detect SARS-CoV-2 virus-encoded proteins from lung tissues obtained at autopsy from patients confirmed to have COVID-19. All samples were collected after prior approval from Mayo Clinic's Institutional Review Board and a written informed consent from the decedent or a family member.

All patients were tested for SARS-CoV-2 virus using the routine RT-PCR testing on nasopharyngeal or oropharyngeal swab samples. Formalin-fixed paraffin-embedded (FFPE) lung tissue sections from six patients were subjected to bottom-up proteomic analysis which included protein extraction followed by overnight trypsin digestion (Fig. 1A). Digested peptides were fractionated using basic pH reversed-phase liquid chromatography followed by LC-MS/MS analysis on a high-resolution mass spectrometer coupled to a nanoLC platform. Database searching was performed against a combined database of human and SARS-CoV-2 viral protein sequences using Sequest search engine. Detailed methods employed for mass spectrometry analysis are provided in Supplementary File S1.

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FIG. 1.

SARS-CoV-2 detection in human lung FFPE tissues using mass spectrometry. (A). Sample preparation workflow for the proteomic analysis of FFPE tissues. Briefly, two 4 μm lung tissue scrolls were deparaffinized followed by rehydration in ethanol and finally in water. Protein extraction was performed using pressure-cycling technology in Barocycler followed by overnight trypsin digestion. Digested peptides were fractionated, and fractions were then subjected to LC-MS/MS analysis. Protein identification was performed using Sequest search engine in Proteome Discoverer 2.3 version. (B) Summary of SARS-CoV-2 virus-derived peptides identified from lung FFPE tissues. The filled boxes indicate that the peptide was identified in the corresponding sample while unfilled boxes indicate peptide not identified. Representative MS/MS spectrum of the SARS-CoV-2 viral peptides identified in the FFPE tissues. (C) DGIIWVATEGALNTPK, (D) AYNVTQAFGR (E) IGMEVTPSGTWLTYTGAIK belong to nucleocapsid protein and (F) VAGDSGFAAYSR belong to membrane protein of SARS-CoV-2 virus. The code given in the parenthesis beside peptide sequence indicates the FFPE tissue sample in which the viral peptide is identified. FFPE, formalin-fixed paraffin-embedded.

From the six FFPE samples tested, we identified a median of nearly 7000 proteins, and nearly 54,000 peptides (Supplementary Table S1). SARS-CoV-2 peptides derived from four different viral proteins (nucleocapsid, membrane glycoprotein, ORF3a, and ORF9b) were identified from four out of six autopsy tissue samples (Fig. 1B). Nucleocapsid protein was identified from all four positive samples owing to its high expression levels in SARS-CoV-2. Representative tandem mass (MS/MS) spectra of viral peptides identified from each sample were shown in Figure 1C–F that includes DGIIWVATEGALNTPK, AYNVTQAFGR, IGMEVTPSGTWLTYTGAIK peptides from nucleocapsid protein and VAGDSGFAAYSR from membrane glycoprotein. The FFPE samples were also tested for presence of viral RNA using RNAScope in situ hybridization technology and ddPCR (Supplementary Table S2).

Of the four samples that were positive for SARS-CoV-2-encoded peptides by mass spectrometry, three samples were positive by both RNAScope and ddPCR. Notably, although several peptides from SARS-CoV-2, that is, three peptides from nucleocapsid protein and one peptide each from membrane glycoprotein and ORF3a, were identified from the fourth sample (S4), it was negative by RNAscope and contained barely detectable viral RNA copies by ddPCR. We did not identify SARS-CoV-2 derived peptides from two of the samples (S5 and S6)—these two samples also tested negative by RNAscope and barely detectable viral RNA as determined by ddPCR.

To our knowledge, this is the first demonstration of peptides detected from lung tissues from patients with COVID-19, although reports employing mass spectrometry-based platforms for detecting viral proteins from nasopharyngeal swabs (Mangalaparthi et al., 2021) and urine samples (Chavan et al., 2021) have been published. Importantly, viral peptides identified using limited material from FFPE tissues as shown in this study might enable opportunities for advancing our understanding of viral infections beyond SARS-CoV-2. Overall, this study and findings highlight the potential of mass spectrometry as a promising method for identification of viral proteins including SARS-CoV-2 or other pathogens from clinical tissue samples.