Kidney transplantation faces a critical paradox: while thousands await organs, approximately 30% of potential deceased donor kidneys are discarded for various reasons, including subjective assessments due to the lack of an objective molecular biomarker of preservation quality. Here, we applied novel “top-down” proteoform imaging mass spectrometry across living donor (LD), deceased donor (brain death or cardiac death), and discarded human kidneys to quantify proteoforms correlating with post-transplant kidney function. This approach preserves post-translational modifications and splice variants, revealing molecular tissue variability beyond protein presence.

LD kidneys displayed robust metabolic signatures, including L-xylulose reductase and cytochrome oxidase subunits, whereas deceased donor and discarded organs showed elevated cellular stress markers such as alpha-B-crystallin and peroxiredoxin 1. Post-transplant blood proteoform analysis validated tissue findings, demonstrating persistent cellular stress and immune activation in deceased donor recipients compared with physiologic wound healing in LD recipients. Consistent with these molecular predictions, serum creatinine levels were highest in DCD, intermediate in DBD, and lowest in LD recipients.

The intersection of tissue proteoform signatures across all marginal tissues identified four proteoforms consistently elevated in deceased and discarded kidneys: ACTG1, acetylated CRYAB, PARK7, and S100A4. Collectively, these proteoforms capture key molecular indicators of graft quality, reflecting oxidative stress, cellular injury, and immune activation pathways. As such, they represent promising point-of-care (POC) biomarker candidates for objective kidney classification, potentially improving donor kidney utilization.

Translational statement Current methods for evaluating donor kidney quality rely on subjective assessments, contributing to the discard of approximately 30% of potentially viable organs. This study demonstrates that “top-down” proteomics can objectively identify molecular signatures distinguishing high-quality from marginal donor kidneys. Top-down proteomics analyzes intact proteins with their post-translational modifications or cleavage products, termed proteoforms to provide mechanistic insights into graft quality. We identified four proteoforms (ACTG1, acetylated CRYAB, PARK7, and S100A4) to be consistently elevated in deceased and discarded kidneys, reflecting oxidative stress, cellular injury, and immune activation. These molecular markers correlated with post-transplant kidney outcome, as measured by serum creatinine levels and recipient blood proteoforms. As a next step, validation in larger cohorts could establish these proteoforms as point-of-care biomarkers for real-time donor kidney assessment during procurement. This objective molecular stratification could reduce unnecessary organ discards and improve transplant outcomes by matching organ quality with recipient risk profiles.

S.N.N. serves as the Chief Medical Advisor for Pandorum Technologies Pvt Ltd. and on the Scientific Advisory Board for TransMedics Inc. N.L.K. reports a conflict of interest with I2MS technology, being commercialized by Thermo Fisher Scientific. N.L.K. is involved in the commercialization of data analysis software and is a paid consultant for Thermo Fisher Scientific.

This work was supported by National Institutes of Health grant AI142079-01A1 to S.N.N. Additional funding was received from Northwestern Medicine Dr. Michael M. Abecassis Transplant Innovation Endowment Grant FY2024 to N.L.K. and E.F. We gratefully acknowledge all members of the Kelleher and Nadig laboratories for their support.

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

The research protocol was approved by Northwestern Medicine Institutional Review Board. Tissue and liquid biopsies were collected in accordance with the Northwestern Medicine Institutional Review Board requirements.

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The mass spectrometry datasets generated for this study will be deposited to MASSIVE (https://massive.ucsd.edu/ProteoSAFe/) and will be made available upon publication.