Delayed Times to Tissue Fixation Result in Unpredictable Global Phosphoproteome Changes

Protein
phosphorylation controls the activity of signal transduction
pathways regulated by kinases and phosphatases. Little is known, however,
about the impact of preanalytical factors, for example, delayed times
to tissue fixation, on global
phosphoprotein levels in tissues. The aim of this study was to characterize
the potential effects of delayed tissue preservation (cold ischemia)
on the levels of phosphoproteins using targeted and nontargeted proteomic
approaches. Rat and murine liver samples were exposed
to different cold ischemic conditions ranging from 10 to 360 min prior
to cryopreservation. The phosphoproteome was analyzed using
reverse phase protein array (RPPA) technology and phosphoprotein-enriched
quantitative tandem mass spectrometry (LC-MS/MS). RPPA analysis of
rat liver tissues with long (up to 360 min) cold ischemia times did
not reveal statistically significant alterations of specific phosphoproteins
even though nonphosphorylated cytokeratin 18 (CK18) showed increased
levels after 360 min of delay
to freezing. Keeping the samples on ice prior to cryopreservation
prevented this effect. LC-MS/MS-based quantification of 1684 phosphorylation
sites in rat liver tissues showed broadening of their distribution
compared to time point zero, but without reaching statistical significance
for individual phosphosites. Similarly, RPPA analysis of mouse liver
tissues with short (<60 min) cold ischemia times did not reveal
directed or predictable
changes of protein and phosphoprotein levels. Using LC-MS/MS and quantification
of 791 phosphorylation sites, we found that the distribution of ratios
compared to time point zero broadens with prolonged ischemia times,
but these were rather undirected and diffuse changes, as we could
not detect significant alterations of individual phosphosites. On
the basis of our results from RPPA and LC-MS/MS analysis of rat and
mouse liver tissues, we conclude that prolonged cold ischemia results
in unspecific phosphoproteome changes that can be neither predicted
nor assigned to individual proteins. On the other hand, we identified
a number of phosphosites which were extraordinarily stable even after
360 min of cold ischemia and, therefore, may be used as general reference
markers for future companion diagnostics for kinase inhibitors.