Amino acid catabolite markers for early prognostication of pneumonia in patients with COVID-19

For IMS, lung tissues from heat-fixed SARS-CoV-2-infected or freshfrozen influenza-infected mice were embedded in super cryoembedding medium (SCEM; SECTION LAB, Hiroshima, Japan) and then

stored at −80 °C until analysis. The tissue-containing SCEM blocks

were sectioned at a cryostat temperature of −16 °C into 8-μm-thick

sections using a CM 3050 cryostat (Leica, Wetzlar, Germany). These

sections were transferred to indium tin oxide (ITO)-coated glass

slides (Matsunami Glass Industries, Osaka, Japan) for further analyses. The mounted sections were manually coated with a matrix

solution containing 9-aminoacridine (10 mg/mL dissolved in 80%

ethanol) using an art brush (Procon Boy FWA Platinum, Mr. Hobby,

Tokyo, Japan). The matrix solution was applied from a distance of

approximately 15 cm, with approximately 1 mL sprayed on each

slide. To maintain uniform conditions for analyte extraction and cocrystallization, matrix application was performed simultaneously

on multiple slides, as previously described60. Optical images of lung

sections were obtained by a scanner and subjected to MALDI-MS

imaging.

7.

IMS

8.

Matrix-assisted laser desorption/ionization (MALDI) imaging was performed using a Bruker timsTOF fleX MS (Bruker Daltonics, Bremen,

Germany) operated in quadrupole time-of-flight (qTOF) analysis mode.

The acquisition parameters were set as follows: negative ion mode

detection, pixel resolution of 80 μm, 200 laser pulses per pixel at a

frequency of 10 kHz, and laser power setting of 50%. Data acquisition

was targeted to an m/z range of 100–650. The raw mass spectra were

processed and reconstructed using SCiLS Lab (v. 2019, Bruker Daltonics), which allowed the generation of detailed MS images. Signals

within the targeted range were normalized to the total ion current to

account for variations in ionization efficiency among pixels. The

imaged metabolites were primarily identified as nucleotides and related molecular species, following previously published protocols55.

Metabolite identifications were validated based on accurate mass

measurements, and congruence checks with reference standards

analyzed by MALDI-MS were used to further confirm the m/z consistency of the identified ions.

1.

2.

3.

4.

5.

6.

9.

10.

11.

12.

13.

Statistical analysis

Statistical analyses were performed using Prism v. 9.5.1 (GraphPad

Software, Inc., San Diego, CA, USA). The analyses included unpaired

Student’s t-test, one-way ANOVA with Tukey’s multiple comparisons

test, and/or two-way ANOVA with Tukey’s multiple comparisons tests,

as specified in each figure legend.

Reporting summary

14.

15.

16.

Further information on research design is available in the Nature

Portfolio Reporting Summary linked to this article.

17.

Data availability

The metabolomics raw data from COVID-19 patients or healthy control

serum have been deposited in Metabolomics Workbench under the

DOI for this project (ST002984): https://doi.org/10.21228/M8DT65. All

data generated or analyzed in this study are available with this article

Nature Communications | (2023)14:8469

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Acknowledgements

This research was supported by the Japan Society for the Promotion of

Science KAKENHI (22K15927 to Y.N.), the Japan Agency for Medical

Research and Development (JP20jk0110021 to Y.N., and JP20he1122001

and JP20wm0125003 to Y.Kido), the Osaka Metropolitan University

Strategic Research Grant (OCU-SRG2021_YR09 to Y.N.), the COVID-19

Private Fund (to Y.Kido), Moonshot Research & Development

(JPMJMS2025 to M. Sasai, M.Y., Y.M., 22zf0127007s0301 to M.S.) and AllOsaka U Research in “The Nippon Foundation - Osaka University Project

16

Article

for Infectious Disease Prevention,” AMED-SCARDA project for Vaccine

Development (Toshio Ito as the Lead) as a collaborator in Central Institute for Experimental Animals (M. Suematsu), the Japan Agency for

Medical Research and Development (23zf0127007s0102,

JP23zf0127003, 22fk0108511h0001 and JP23gm1210009 to Y.S.), JSPS

KAKENHI (22H02833 to Y.S.).

Author contributions

R.M., N.T., and Y.S. performed the metabolome and histological analysis.

N.K.S., M.M., M.Su., M.Yan., and Y.S. wrote the manuscript. Y.U. M.W. Y.N.

and Y.Kido collected samples for a clinical study. M.Sa., M.Yam., Y.M.

J.U., G.Y., M.H., S.T., and Y.Kim performed the infection experiment. Y.S.

conceptualized the study and provided methodology and investigation.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains

supplementary material available at

https://doi.org/10.1038/s41467-023-44266-z.

Correspondence and requests for materials should be addressed to

Yuki Sugiura.

Nature Communications | (2023)14:8469

https://doi.org/10.1038/s41467-023-44266-z

Peer review information Nature Communications thanks Katharina Kurz,

Slobodan Paessler, and the other, anonymous, reviewer(s) for their

contribution to the peer review of this work.

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