Aims/Hypothesis Multiple studies associated enterovirus (EV) infections with type 1 diabetes. The Network for Pancreatic Organ Donors with Diabetes (nPOD) obtained samples from organ donors with/without type 1 diabetes and launched the nPOD-Virus Group to examine viral infections in donor tissues, using complementary approaches. To this end, we aimed to identify virus proteins/peptides in disease-stratified tissues using proteomic and liquid chromatography-mass spectrometry. Methods nPOD provided specimens from four donor groups: donors without diabetes (ND, n=33), with type 1 diabetes (T1D, n=25), with type 2 diabetes (T2D, n=7), and without diabetes expressing type 1 diabetes-associated autoantibodies (AAb+, n=17; preclinical disease). We studied flash-frozen pancreas tissue chunks, embedded tissue slices, and islets obtained via laser capture microdissection (LCM). We isolated and processed proteins from these specimens for liquid chromatography-mass spectrometry analysis. We utilized different instruments including a Q-Exactive Orbitrap Mass spectrometer and an Orbitrap Fusion Lumos Mass Spectrometer to acquire high resolution, high mass accuracy and high sensitivity MS data using different scanning methods. We used data dependent acquisition (DDA), data independent acquisition (DIA), and parallel reaction monitoring (PRM). Generated mass spectra were processed and used in protein database searches for identification, qualitative and quantitative comparative analyses of viral protein expression in tissue samples. Results Advanced proteomics were applied to pancreata from 82 disease-stratified nPOD donors. These analyses generated >1,000 individual mass spectra data files. We identified enterovirus peptides from different serotypes in 28 donors, including 11 donors with type 1 diabetes. These serotypes included several previously associated with type 1 diabetes. For some donors, identification of virus peptides by discovery proteomics was validated by targeted mass spectrometry and Western blot. Conclusions/Interpretation For the first time we applied complementary mass spectrometry-based proteomics to detect viral proteins in disease-stratified pancreas samples. Some pancreata, including several from donors with type 1 diabetes, were infected by enteroviruses based on detection of viral proteins; in several instances we identified serotypes, which has been arduous with other methods. We detected both structural and non-structural viral proteins, the latter essential for replication, suggesting that enteroviruses may replicate in pancreas, perhaps at low level, given the absence of acute infection. The complexity of our methodology limited application to large sample sets, and accordingly we did not aim to demonstrate an association with disease; our data complement associative data generated with other approaches by the nPOD-Virus Group, overall supporting a role for enterovirus infections in type 1 diabetes.

The authors have declared no competing interest.

This research was performed with the support of the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project supported by grants from JDRF/ The Leona M. & Harry B. Helmsley Charitable Trust (3-SRA-2023-1417-S-B) and the Helmsley Charitable Trust (2018PG-T1D053, G-2108-04793). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at https://npod.org/for-partners/npod-partners/. The nPOD-Virus group was supported by JDRF grants (3-SRA-25-2012-516 and 3-SRA-2017-492-A-N awarded to A.P.).

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

University of Florida Health Center Institutional Review Board (IRB Protocol #201600029) The study of de-identified organ donor tissues is considered not to involve human subjects and is exempted from review by the Eastern Virginia Medical School Institutional Review Board (IRB)

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All data produced in the present study are available upon reasonable request to the authors