Delayed times to tissue fixation result in unpredictable global phosphoproteome changes

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Protein phosphorylation is crucial for signal transduction.
• The impact of preanalytical factors like cold ischemia on phosphoprotein levels is largely unknown.
• Understanding these effects is vital for accurate proteomic analysis.

Purpose of the Study:

• To investigate the effects of cold ischemia on phosphoprotein levels in liver tissues.
• To characterize potential alterations in the phosphoproteome due to delayed tissue preservation.
• To identify stable phosphosites for use as reference markers.

Main Methods:

• Rat and mouse liver samples were subjected to varying cold ischemia times (10-360 min).
• Phosphoproteome analysis was performed using reverse phase protein array (RPPA) and quantitative tandem mass spectrometry (LC-MS/MS).
• Statistical analysis was used to assess changes in phosphoprotein levels and phosphorylation sites.

Main Results:

• Prolonged cold ischemia (up to 360 min) in rat liver led to unspecific phosphoproteome changes, not statistically significant for individual sites.
• Nonphosphorylated cytokeratin 18 (CK18) levels increased after 360 min ischemia, an effect prevented by keeping samples on ice.
• Short cold ischemia (<60 min) in mouse liver showed no directed changes; prolonged ischemia broadened phosphosite distribution without significance.
• A subset of phosphosites demonstrated remarkable stability even after extended cold ischemia.

Conclusions:

• Prolonged cold ischemia induces unspecific and unpredictable changes in the phosphoproteome.
• Specific phosphosites exhibit high stability and can serve as reliable reference markers.
• These stable phosphosites hold potential for companion diagnostics in kinase inhibitor therapies.