Ultra-deep longitudinal profiling of plasma and serum proteome provides critical insights into the interaction of human organism and pig xenotransplants

Xenotransplantation of pig organs to humans holds immense potential to address the critical shortage of donor organs. Recent advancements in genetic engineering and immunosuppressive therapies have brought xenotransplantation closer to reality, with successful pig-to-human kidney and heart transplants marking significant milestones. However, understanding the complex molecular interactions between the recipient's organism and the xenograft remains a major challenge. Measuring plasma proteome of these patients provides critical information about immune response and status of many organism subsystems.

In a series of studies, we employed an advanced proteomic workflow, combining nanoparticle-based sample preparation (Proteograph™ XT, Seer, Inc.) with the state-of-the-art LC-MS using Ultimate™ 3000 HPLC and Thermo Scientific™ Orbitrap™ Astral™ MS. Both components allowed us to significantly improve the proteome coverage in comparison to the standard workflows and deliver ultra-deep profiling of plasma or serum proteomes in human recipients of a genetically modified pig organ, including the unprecedented daily serum monitoring of the brain-dead patient with xenokidney over 61 days.

The samples were analyzed in a DIA mode with 3 m/z isolation window using a 30-min gradient. In these datasets we identified around 100K peptides, half of which could be specifically assigned to human or pig proteins and allowed us to reconstruct around 6,200 human and 1,000 pig protein groups. Our analysis revealed the intricate regulation patterns for thousands of human and pig proteins. We observed significant changes in the complement system, cytokines, immunoglobulins, and various metabolic pathways, reflecting the recipient's immune response and the physiological adaptation to the xenograft. Notably, we could track the abundance dynamics for hundreds of pig-specific proteins in the recipient's blood, highlighting the molecular details of xenograft integration, including its immune system response.

By using the whole plasma proteome as a comprehensive indicator of a recipient"s state, we could observe the differential dynamics of individual xenotransplantation patients that correlated with the other clinical and omics data. Such analysis is instrumental in identifying potential human and xenograft biomarkers that would contribute to more efficient post-operational treatment of these patients. Ultimately, this research paves the way for xenotransplantation to become a viable solution for patients in need of life-saving organ transplants.