Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions.

PURPOSE: The organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient. The objective of this study was to assess two bottom-up proteomics workflows for the extraction of tryptic peptides from the perfusate. EXPERIMENTAL DESIGN: Two different kinds of perfusate samples from kidney and liver trials were profiled using liquid chromatography-mass spectrometry (LC-MS/MS). The preparation of clean peptide mixtures for downstream analysis was performed considering different aspects of sample preparation; protein estimation, enrichment, in-gel and urea-based in-solution digestion. RESULTS: In-solution digestion of perfusate allowed identification of the highest number of peptides and proteins with greater sequence coverage and higher confidence data in kidney and liver perfusate. Key pathways identified by gene ontology analysis included complement, coagulation and antioxidant pathways, and a number of biomarkers previously linked to ischemia-reperfusion injury were also observed in perfusate. CONCLUSIONS: This study showed that in-solution digestion is a more efficient method for LC-MS/MS analysis of kidney and liver organ perfusion solutions. This method is also quicker and easier than in-gel digestion, allowing for greater sample throughput, with fewer opportunities for experimental error or peptide loss.

Assessment of Mitochondrial Function and Oxygen Consumption Measured During Ex Vivo Normothermic Machine Perfusion of Injured Pig Kidneys Helps to Monitor Organ Viability.

Donor kidney assessment may improve organ utilisation. Normothermic Machine Perfusion (NMP) has the potential to facilitate this advance. The mechanism of action is not yet determined and we aimed to assess mitochondrial function during NMP. Anaesthetised pigs (n = 6) had one kidney clamped for 60 min. The healthy contralateral kidney was removed and underwent NMP for 8 h (healthy control (HC), n = 6). Following 60 min warm ischaemia the injured kidney underwent HMP for 24 h, followed by NMP for 8 h (n = 6). Mitochondria were extracted from fresh tissue for analysis. Injured kidneys were analysed as two separate groups (IMa, n = 3 and IMb, n = 3). Renal resistance was higher (0.39ï, ± 0.29 vs. 1.65ï, ± 0.85; p = 0.01) and flow was lower (55ï, ± 28 vs. 7ï, ± 4; p = 0.03) during HMP in IMb than IMa. NMP blood flow was higher in IMa versus IMb (2-way ANOVA; p < 0.001) After 60 min NMP, O2 consumption was significantly lower in IMb versus IMa (p ≤ 0.002). State-3 respiration was significantly different between the groups (37ï, ± 19 vs. 24ï, ± 14 vs. 10ï, ± 8; nmolO2/min/mg; p = 0.049). Lactate levels were significantly lower in IMa versus IMb (p = 0.028). Mitochondrial respiration levels during NMP may be suggestive of kidney viability. Oxygen consumption, renal blood flow and lactate can differentiate severity of kidney injury during NMP.