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 w...

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Bibliographic Details
Published in:Journal of proteome research 2013-10, Vol.12 (10), p.4424-4434
Main Authors: Gùˆndisch, Sibylle, Grundner-Culemann, Kathrin, Wolff, Claudia, Schott, Christina, Reischauer, Bilge, Machatti, Manuela, Groelz, Daniel, Schaab, Christoph, Tebbe, Andreas, Becker, Karl-Friedrich
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Cell Hypoxia
Cell Line, Tumor
Chromatography, Reverse-Phase
Cryopreservation
Liver
Mice
Mice, Inbred C57BL
Molecular Sequence Data
Peptide Fragments - chemistry
Phosphoproteins - chemistry
Phosphoproteins - metabolism
Phosphorylation
Protein Kinases - metabolism
Protein Processing, Post-Translational
Proteome - chemistry
Proteome - metabolism
Rats
Rats, Wistar
Reference Standards
Tandem Mass Spectrometry - standards
Time Factors
Tissue Fixation
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Summary: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 (
ISSN:1535-3893
1535-3907
DOI:10.1021/pr400451z