Introduction: Hepatic ischemia/reperfusion injury (IRI) is a major risk factor for acute rejection and early graft dysfunction in liver transplantation. The large and complex landscape of kinase driven regulatory signaling events in IRI is not well characterized. To understand the hepatic phosphoproteomic regulatory changes during the early stages of IRI, we used TMT Isobaric Tags for Relative and Absolute Quantitation-based analyses in a mouse hepatic IRI model.

Methods: C57BL/6 mice were subjected to 70% partial warm ischemia for 90min, followed by 15m, 2h, or 6h of reperfusion. Naive mice were included to establish a baseline. Hepatic tissues from euthanized mice were homogenized, trypsin digested, and labeled with isobaric reagents to provide multiplex relative quantitation of individual samples. Samples were fractionated and enriched for phosphopeptides using IMAC (immobilized metal affinity chromatography). Flow-through fractions were subjected to global proteomic analysis; phosphopeptides were further fractionated and then resolved on a quadropole-orbitrap mass spectrometer in tandem with nLC-MSMS. Raw data were analyzed in Proteome Discoverer 2.2.

Results: Using this method more than 15,000 events were measured. A large number of the identified pathways have previously been identified as potential drug targets to inhibit IRI. However, many kinases have not yet been examined in this context. Several characteristic patterns emerged in the measured phosphoprotein levels and related kinase activities. Apoptosis pathways, including PAK2, are highly active in ischemic tissues and then their activity decreases upon reperfusion (A). Tyrosine kinases, such as Syk, ZAP70, Blk, and Lyn, demonstrate increased activity as early as 15 mins after reperfusion and maintain this activity up to 6 hrs after reperfusion (B). After 2 hrs, MAP kinases such as p38a and JAK2/3 and other kinases involved in immune cell activation peak in activity and stay active up to 6 hrs after reperfusion paralleling cellular infiltration (C).

Conclusion: This study identifies previously unexamined pathways important for IRI and presents potential targets for drug therapy.

CITATION INFORMATION: Zarrinpar A., Duarte S., Lai Y. Phosphoproteomic Analysis of Mouse Hepatic Ischemia/Reperfusion Injury Am J Transplant. 2017;17 (suppl 3).

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