Untargeted metabolomic profiling of sera from AMN-SARS-CoV-2 cases
Each serum sample underwent meticulous processing and analysis using UHPLC-OE-MS for untargeted metabolomics, following a standardized protocol. This approach encompassed the examination of both hydrophilic and hydrophobic molecules via positive and negative ionization modes, thereby ensuring a comprehensive assessment of diverse endogenous biochemical classes. The analysis revealed a total of 15,585 metabolite peaks, including 7,182 metabolites in the negative ion mode and 8,403 differential metabolites in the positive ion mode. Quality control (QC) samples exhibited consistency within ± 2 standard deviations, thus validating the precision, reliability, and consistent outcomes of the experimental method employed. OPLS-DA plots visually depicted the distinct clustering of AMN-SARS-CoV-2 and SARS-CoV-2-no AMN groups in both discovery and validation cohorts (Figure 1A). Subsequent analysis led to the identification of 498 metabolites through MS/MS spectra based on the MS2 database (BiotreeDB), KEGG, and HMDB databases. Of these, 118 metabolites were significantly correlated with AMN-SARS-CoV-2 (Figure 1B; p < 0.05; VIP > 1). A volcano plot was generated, revealing 76 significantly upregulated metabolites and 42 downregulated metabolites in the AMN-SARS-CoV-2 group compared to the SARS-CoV-2-no AMN group (Figure 1C). Notably, several amino acids exhibited marked changes among these significant metabolites. Specifically, the AMN-SARS-CoV-2 group displayed significant upregulation of L-asparagine, L-serine, L-pyroglutamic acid, glycine, asymmetric-dimethylarginine (ADMA), ornithine, D-ornithine, L-proline, sarcosine, L-threonine, citrulline, D-serine, N-acetyl-L-alanine, N-acetyl-L-methionine, L-lysine, L-beta-aspartyl-L-threonine, prolylhydroxyproline, and N-acetylserine (Figure 1D). Conversely, leucyl-isoleucine, alanyl-leucine, serylalanine, leucyl-phenylalanine, phenylalanylphenylalanine, and phenylalanyl-glycine levels demonstrated significant downregulation (Figure 1E).