Delayed Times to Tissue Fixation Result in Unpredictable Global Phosphoproteome Changes

Abstract

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.

Ischemia in Tumors Induces Early and Sustained Phosphorylation Changes in Stress Kinase Pathways but Does Not Affect Global Protein Levels

“…The effects observed in our study were larger in amplitude and did not return to baseline, possibly because of tissuespecific differences or the longer total ischemia time in the RPPA study. In a recent LC-MS/MS-based analysis of delayed freezing of mouse and rat livers, Gundisch et al found that most phosphoproteome changes are diffuse and unpredictable and can be minimized when samples are kept on ice prior to fixation (34). The lack of a common cold-ischemia response in this study may be explained by different stressresponse mechanisms in healthy and diseased tissues.…”

“…Hennessy et al analyzed the effects of cold ischemia in breast cancer tumor samples using RPPA and found that 21 of 82 proteins and phosphoproteins demonstrated time-dependent instability at room temperature between 6 and 24 h of cold ischemia but exhibited few effects at earlier time points (9). Gundish et al used both RPPA and LC-MS/MS to analyze a time course of cold ischemia in normal liver tissue from mouse and rat (34). No significant changes were observed in the phosphoproteome after up to 60 min of cold ischemia, in agreement with the findings of Hennessy et al In the LC-MS/MS study by Gundisch et al, approximately 1700 phosphosites were quantified, but no statistically significant alterations of individual phosphosites after over 60 min of cold ischemia were found.…”

Phosphotyrosine Signaling Analysis in Human Tumors Is Confounded by Systemic Ischemia-Driven Artifacts and Intra-Specimen Heterogeneity

“…Freezing delays following sample resection and processing subject the specimen to ischemia; unfortunately the exact time to freezing for archived tissue specimens is often undocumented. To date, studies on the effects of ischemia on tissue phosphorylation have been reported, yet the sample collection and analysis approaches used did not allow assessment of short periods of ischemia (13) or permit comprehensive analysis of pTyr signaling (14). …”

Thermal Inactivation of Enzymes and Pathogens in Biosamples for MS Analysis

“…In the search for biomarkers, a criterion should be that they are not subject to change in the pre-analytical phase [17,18]. Preanalytical degradation limits the discovery of potential biomarkers.…”