The epithelial barrier's primary role is to protect against entry of foreign and pathogenic elements. Global and targeted approaches were applied to nasal swabs from healthy and COVID-19-confirmed cases within 24 hours post-positive-confirmation and at 3 weeks post-infection to observe changes in proteome and metabolome.

We found that the tryptophan/kynurenine metabolism pathway is a pinch-point regulator of canonical and non-canonical transcription activation, macrophage release of cytokines and significant changes in the immune and metabolic status with increasing severity and disease course.

Nasal epithelial swabs were self-collected by participants in this study. Swabs were resuspended in 80% methanol with 6mg of 1.0 mm zirconium beads and used cell shearing to extract proteins and metabolites. The method is described in Wasinger et al., 2020 [1]. Proteins were pelleted and the supernatant containing metabolites stored at -80°C until required. Protein pellet was resuspended in digestion buffer and 50 µg enzymatically treated with trypsin overnight at room temperature.

Proteomic mass spectrometry

Mass spectrometry was carried out using a QExactive (Thermo Electron, Bremen, Germany)  run in DDA mode using 1.5 μg (2.0 μL from 10μL) as previously described [2]. Peptides were eluted using a linear gradient of H₂O:CH₃CN (98:2, 0.1% formic acid) to H₂O:CH₃CN (64:36, 0.1% formic acid) at 250 nL min^-1 over 60 min.

Statistical Analysis

Proteins were identified using Mascot Daemon v2.5.1 (Matrix Science, London, UK) searched against the SwissProt and SARV19 database (downloaded February 2021, containing 563,972 sequences; and July 2020, containing 271,909 sequences, respectively). Search parameters were set to carbamidomethyl (C); variable modifications, oxidation (M), phospho (STY); enzyme, semi-Trypsin; and maximum missed cleavages, 1; peptide tolerance, ± 5 ppm; fragment tolerance, 0.05 Da. Scaffold software (version 4.6.1, Proteome Software Inc., Portland, OR, USA) was used to compare the proteome. Peptide identifications were accepted if they could be established at greater than 95% probability using the Scaffold delta-mass correction. Protein identifications were accepted if they could be established at less than 1% false discovery rate (FDR) and contained at least 2 identified peptides. Expression changes across the samples were measured via spectral count, normalised to total ion count. ANOVA was used to report abundance changes controlled by the Benjamini-Hochberg procedure for multiple comparisons, with p-values set to <0.05. The studies reached a power ≥ 90% and were calculated using PASS software based on a mean abundance values and standard deviation between groups.

The proteomic dataset of differentially abundant proteins was assessed for enriched pathways using Ingenuity Pathway Analysis (IPA® Qiagen, CA, USA). The core analysis was carried out using the default settings with only direct relationships and only experimentally observed confidence considered based on the IPA knowledge base (genes only). The P-value for the correlation between identified proteins and a given canonical pathway was calculated by Fisher's exact test.

Targeted proteins were analysed using Skyline Software, and peptides were accepted based on retention time and sequence with at least 3 transitions required. Peak area under curve of the parent ion was used to assess relative abundance of the marker panel. Log₂ transformed data were evaluated using Student T-test, and Receiver Operating Characteristic (ROC) probability curves to measure ability to distinguish between binary classifiersPRM targeted analysis applied transitions listed in Attachment.

Quantification of Kynurenine Pathway

Mixed standards and 100 µl aliquots of Nasal methanolic extracts were spiked with an internal standard mixture containing labelled KP metabolites; dried, and reconstituted in 100 µl of water, filtered through 4 mm syringe filters with 0.2 μm membrane into reduced volume LC vials; 20 µl aliquots were injected for analysis. 

MRM LC-MS/MS analysis was conducted using a TSQ Vantage mass spectrometer (Thermo, USA) connected to Vanquish (Thermo-Dionex USA) solvent delivery/autosampler system. Chromatographic separation was achieved using a Kinetex™ PFP column (150mm x 2 mm, 1.7μm, 100 Å, Phenomenex USA) by reverse phase gradient elution at 25˚C using a gradient of 0.1% formic acid to 10% methanol over 2 min, then ramped to 60% B to 4min, and then ramped to 100%B by 8mins.

Quantification of NAD+ome metabolites

LC-MS/MS analysis was conducted using a TSQ Vantage mass spectrometer (Thermo, USA) connected to Vanquish (Thermo-Dionex USA) solvent delivery/autosampler system. Chromatographic separation was achieved using a Kinetex™ PFP column (150mm x 2 mm, 1.7μm, 100 Å, Phenomenex USA) by reverse phase gradient elution at 25˚C. The mobile phase consisted of aqueous 0.1% formic acid (A) and methanol (B). The gradient elution was programmed as follows: start at 10 % B, hold 2 minutes, ramp to 60%B in 4min, then to 100%B in 8min. In 0.4min set to 10 % B and equilibrate for 5.6 min. Total run time is 20 min. 

Mass spectrometric detection was performed using multiple reaction monitoring (MRM) with heated electrospray ionization (HESI) source in positive mode. MSD parameters were optimised using Anthranilic acid direct infusion, and the tune file created was used in the created method. The conditions were: ion spray voltage, 4,000 V; vaporizer temperature 300˚C, capillary temperature 300˚C, collision argon gas 1 Torr, sheath and auxiliary gas valves (nitrogen) set at 20 and 10 arbitrary units respectively. The MRM transitions for all analytes were optimised using a syringe infusion pump and are shown in Attachment 1. Data acquisition and processing were performed with Xcalibur™ (version 2.2, 2011 Thermo Fischer Scientific, Waltham MA).

NAD+ome LCMS/MS assay of nasal epithelial (NE) swab extracts

Methods followed Bustamante et al. [3]. LC-MS/MS analysis was conducted using a TSQ Vantage mass spectrometer (Thermo, USA) connected to Vanquish (Thermo-Dionex, USA) solvent delivery system/autosampler using an adaptation of a previously published method by Bustamante et al. [5]. Isotopically enriched (2H) internal standards were purchased from Toronto Research Chemicals and primary standards from Sigma-Aldrich. HESI-MS parameters: Ion spray voltage 4,000 V; vaporizer temperature 300˚C, capillary temperature 300˚C, collision gas 1.0 Torr. These parameters were optimised using NMN solution in positive ion mode. Calibrators of known concentrations (0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4 μM) of NADOME metabolites were prepared by mixing aliquots of standards with a fixed volume of internal standard mixture. Similarly, NE extracts were mixed with internal std. cocktail, dried and reconstituted in 50 µl of 100 mM ammonium acetate in water. Samples were filtered into LC vials and 20μL injected for analysis. Data acquisition and processing were performed with Xcalibur™ (version 2.2, 2011 Thermo Fischer Scientific, Waltham MA). Mobile phases consisted of 5mM ammonium acetate in water pH 9.5 (A); 100 % Acetonitrile (B) according to Table S5 using a Phenomenex Luna 3 µm NH2 100 Å 150 x 2 mm column.

Racemic amino acid analysis

Methods were adapted from Ayon et al. [4]. Briefly, 40 µl of colon biopsies extracts were mixed with ²H₄-alanine as internal standard. Samples were dried and derivatised with 20 µl of 10mM Marfey’s reagent in acetone and 5 µl of triethylamine and incubated at 37˚C for 3 hours, the reaction was quenched with 10 µl 0.5 M HCl. Samples were diluted with 120 µl of 30 % ACN in 0.1% aqueous formic acid. Phenomenex SPE Strata-X cartridges (30 mg) were preconditioned with methanol, followed by 0.1 % formic acid in water, and samples were loaded and washed with 0.1 % formic acid in water, and then eluted with 70 % acetonitrile in 0.1 % aqueous formic acid. Eluants were dried and reconstituted in 0.1 % aqueous formic acid before analysis.  LC-MS/MS analysis was conducted using a TSQ Vantage mass spectrometer as described in Attachment 1.

GCMS/MS assay of nasal epithelial (NE) swabs of picolinic and quinolinic acid

GC-MS analysis was carried out using Agilent Technologies GCMS system comprising 5973inert MSD coupled to 6890 GC oven and 7683 series autosampler. Chromatographic column Agilent J&W DB5-MS UI 30mx 0.25mm x 0.25μm. Methods followed those described by Smythe et al. [5].

Single Ion Monitoring (SIM) GC-MS assay of picolinic and quinolinic acid in nasal swab extracts. 

Picolinic and quinolinic acid in NE extracts were assayed by GC–MS in electron-capture negative ionization mode; a very sensitive method with on-column limit of detection for QUIN and PIC < 1 femtomol on column (Smythe et al. 2003). Briefly, standards and NS extracts (100-200μl) were spiked with ²H₄ -Pic and ²H₃-Quin in 13x100mm glass cell culture tubes, and dried in a Speedvac before derivatisation with 60μL TFAA and 60μL of HFP. Capped tubes were placed in a heating block at at 60°C for 30 min to produce the hexafluoro-isopropyl esters of the respective acids.  Samples were then dissolved in 80μl of toluene, washed with 1ml of 5% sodium bicarbonate and 1ml of water to remove by-products. The upper toluene layer was passed through anhydrous sodium sulphate mini columns (approx. 500 mg) into autosampler vials, and 2μl were injected into the GC/MS system. Sample concentrations of Pic and Quin were calculated from the standard curves generated.  

Monitored SIM ions for ²H₄ -Pic, Pic, ²H₃-Quin and Quin are m/z 277, m/z 273, m/z 467 and m/z 470 respectively.  

Injector temperature 250˚C, transfer line temperature 280˚C; run time 15.2 minutes using T program below: 

GC-MS analysis was carried out using Agilent Technologies GCMS system comprising 5973inert MSD coupled to 6890 GC oven and 7683 series autosampler. Chromatographic column Agilent J&W DB5-MS UI 30mx 0.25mm x 0.25μm. 

Single Ion Monitoring (SIM) GC-MS assay of picolinic and quinolinic acid in nasal swab extracts

Picolinic and quinolinic acid in NS extracts were assayed by GC–MS in electron-capture negative ionization mode; a very sensitive method with on-column limit of detection for QUIN and PIC < 1 femtomol on column (Smythe et al. 2003). Briefly, standards and NS extracts (100-200μl) were spiked with ²H₄ -Pic and ²H₃-Quin in 13x100mm glass cell culture tubes, and dried in a Speedvac before derivatisation with 60μL TFAA and 60μL of HFP. Capped tubes were placed in a heating block at 60°C for 30 min to produce the hexafluoro-isopropyl esters of the respective acids.  Samples were then dissolved in 80μl of toluene, washed with 1ml of 5% sodium bicarbonate and 1ml of water to remove by-products. The upper toluene layer was passed through anhydrous sodium sulphate mini columns (approx. 500 mg), into autosampler vials, 2μl were injected into the GC/MS system. Sample concentrations of Pic and Quin were calculated from the standard curves generated.  

Monitored SIM ions for ²H₄ -Pic, Pic, ²H₃-Quin and Quin are m/z 277, m/z 273, m/z 467 and m/z 470 respectively.  

Injector temperature 250˚C, transfer line temperature 280˚C; run time 15.2 minutes using T program below:

References

1) V.C. Wasinger, D. Curnoe, C. Boel, N. Machin, H.M. Goh, The Molecular Floodgates of Stress-Induced Senescence Reveal Translation, Signalling and Protein Activity Central to the Post-Mortem Proteome, International journal of molecular sciences 21(17) (2020).

2) V.C. Wasinger, D.; Boel, C.; Machin, N.; Goh, H.M. , The Molecular Floodgates of Stress-Induced Senescence Reveal Translation, Signalling and Protein Activity Central to the Post-Mortem Proteome. , Int. J. Mol. Sci. 21 (2020) 6422.

3) S. Bustamante, Jayasena, T., Richani, D., Gilchrist, R.B., Wu, L.E., Sinclair, D.A., Sachdev, P.S., Braidy, N., Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach. , Metabolomics 14(1) (2017) 15.

4) N.J. Ayon, Sharma, A.D., Gutheil, W.G., LC-MS/MS-Based Separation and Quantification of Marfey's Reagent Derivatized Proteinogenic Amino Acid DL-Stereoisomers., J Am Soc Mass Spectrom 30(3) (2019 ) 448-458.

5) Smythe GA, Poljak A, Bustamante S, Braga O, Maxwell A, Grant R, Sachdev P. ECNI GC-MS analysis of picolinic and quinolinic acids and their amides in human plasma, CSF, and brain tissue. Adv Exp Med Biol. 2003;527:705-12.

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