Proteomic and mass spectrometry-based identification of viral proteins in human tissue samples from the Network for Pancreatic Organ Donors with Diabetes.

Aims/HypothesisMultiple 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. MethodsnPOD 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. ResultsAdvanced 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/InterpretationFor 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. Research in ContextO_ST_ABSWhat is already known about this subject?C_ST_ABSThere are previous studies that examined an association of enterovirus infections with type 1 diabetes by examining pancreas tissue, but those are largely limited to the assessment of a single viral antigen by immunohistochemistry in pancreas tissues obtained at autopsy from recent onset patients, from either an historical archive, or from a small series of biopsies. What is the key question?To examine viral infections in the pancreas from the largest collection of organ donors and disease duration using unbiased comprehensive proteomic and liquid chromatography-mass spectrometry methods. What are the new findings?We present robust mass spectrometry based proteomics data that are validated by complementary western blot results identifying multiple virus proteins from different virus serotypes in pancreas from disease stratified organ donors. Overall, the findings support the existence of chronic or recurrent infections in the pancreas of some patients with type 1 diabetes. How might this impact on clinical practice in the foreseeable future?These results provide strong rationale for advancing current efforts to prevent or mitigate type 1 diabetes by vaccination and/or anti-viral therapies.