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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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| Published in: | Journal of proteome research 2013-10, Vol.12 (10), p.4424-4434 |
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| container_title | Journal of proteome research |
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| creator | Gündisch, Sibylle Grundner-Culemann, Kathrin Wolff, Claudia Schott, Christina Reischauer, Bilge Machatti, Manuela Groelz, Daniel Schaab, Christoph Tebbe, Andreas Becker, Karl-Friedrich |
| description | 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 ( |
| doi_str_mv | 10.1021/pr400451z |
| format | article |
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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.</description><identifier>ISSN: 1535-3893</identifier><identifier>EISSN: 1535-3907</identifier><identifier>DOI: 10.1021/pr400451z</identifier><identifier>PMID: 23984901</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>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</subject><ispartof>Journal of proteome research, 2013-10, Vol.12 (10), p.4424-4434</ispartof><rights>Copyright © 2013 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a381t-98af89ccfeddee88a6261931d0d437212728a039d38ded1533dc1ecbd699c1ff3</citedby><cites>FETCH-LOGICAL-a381t-98af89ccfeddee88a6261931d0d437212728a039d38ded1533dc1ecbd699c1ff3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,777,781,27905,27906</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23984901$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gündisch, Sibylle</creatorcontrib><creatorcontrib>Grundner-Culemann, Kathrin</creatorcontrib><creatorcontrib>Wolff, Claudia</creatorcontrib><creatorcontrib>Schott, Christina</creatorcontrib><creatorcontrib>Reischauer, Bilge</creatorcontrib><creatorcontrib>Machatti, Manuela</creatorcontrib><creatorcontrib>Groelz, Daniel</creatorcontrib><creatorcontrib>Schaab, Christoph</creatorcontrib><creatorcontrib>Tebbe, Andreas</creatorcontrib><creatorcontrib>Becker, Karl-Friedrich</creatorcontrib><title>Delayed Times to Tissue Fixation Result in Unpredictable Global Phosphoproteome Changes</title><title>Journal of proteome research</title><addtitle>J. Proteome Res</addtitle><description>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.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Cell Hypoxia</subject><subject>Cell Line, Tumor</subject><subject>Chromatography, Reverse-Phase</subject><subject>Cryopreservation</subject><subject>Liver</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Molecular Sequence Data</subject><subject>Peptide Fragments - chemistry</subject><subject>Phosphoproteins - chemistry</subject><subject>Phosphoproteins - metabolism</subject><subject>Phosphorylation</subject><subject>Protein Kinases - metabolism</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteome - chemistry</subject><subject>Proteome - metabolism</subject><subject>Rats</subject><subject>Rats, Wistar</subject><subject>Reference Standards</subject><subject>Tandem Mass Spectrometry - standards</subject><subject>Time Factors</subject><subject>Tissue Fixation</subject><issn>1535-3893</issn><issn>1535-3907</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><recordid>eNptkE1Lw0AURQdRbK0u_AMyG0EX0XmZpJlZSrVVKCjS4jJMZl5sSpKJMwlYf72Rfqxcvbs4HN69hFwCuwMWwn3jIsaiGH6OyBBiHgdcsuR4n4XkA3Lm_ZoxiBPGT8kg5FJEksGQfDxiqTZo6KKo0NPW9sH7Dum0-FZtYWv6jr4rW1rUdFk3Dk2hW5WVSGelzVRJ31bWNyvbONuirZBOVqr-RH9OTnJVerzY3RFZTp8Wk-dg_jp7mTzMA8UFtIEUKhdS6xyNQRRCjcMxSA6GmYgnIYRJKBTj0nBh0PR9uNGAOjNjKTXkOR-Rm623f-CrQ9-mVeE1lqWq0XY-hSjiXCYJ8B693aLaWe8d5mnjikq5TQos_dsxPezYs1c7bZdVaA7kfrgeuN4CSvt0bTtX9y3_Ef0CQWh6dg</recordid><startdate>20131004</startdate><enddate>20131004</enddate><creator>Gündisch, Sibylle</creator><creator>Grundner-Culemann, Kathrin</creator><creator>Wolff, Claudia</creator><creator>Schott, Christina</creator><creator>Reischauer, Bilge</creator><creator>Machatti, Manuela</creator><creator>Groelz, Daniel</creator><creator>Schaab, Christoph</creator><creator>Tebbe, Andreas</creator><creator>Becker, Karl-Friedrich</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20131004</creationdate><title>Delayed Times to Tissue Fixation Result in Unpredictable Global Phosphoproteome Changes</title><author>Gündisch, Sibylle ; Grundner-Culemann, Kathrin ; Wolff, Claudia ; Schott, Christina ; Reischauer, Bilge ; Machatti, Manuela ; Groelz, Daniel ; Schaab, Christoph ; Tebbe, Andreas ; Becker, Karl-Friedrich</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a381t-98af89ccfeddee88a6261931d0d437212728a039d38ded1533dc1ecbd699c1ff3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Cell Hypoxia</topic><topic>Cell Line, Tumor</topic><topic>Chromatography, Reverse-Phase</topic><topic>Cryopreservation</topic><topic>Liver</topic><topic>Mice</topic><topic>Mice, Inbred C57BL</topic><topic>Molecular Sequence Data</topic><topic>Peptide Fragments - chemistry</topic><topic>Phosphoproteins - chemistry</topic><topic>Phosphoproteins - metabolism</topic><topic>Phosphorylation</topic><topic>Protein Kinases - metabolism</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteome - chemistry</topic><topic>Proteome - metabolism</topic><topic>Rats</topic><topic>Rats, Wistar</topic><topic>Reference Standards</topic><topic>Tandem Mass Spectrometry - standards</topic><topic>Time Factors</topic><topic>Tissue Fixation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gündisch, Sibylle</creatorcontrib><creatorcontrib>Grundner-Culemann, Kathrin</creatorcontrib><creatorcontrib>Wolff, Claudia</creatorcontrib><creatorcontrib>Schott, Christina</creatorcontrib><creatorcontrib>Reischauer, Bilge</creatorcontrib><creatorcontrib>Machatti, Manuela</creatorcontrib><creatorcontrib>Groelz, Daniel</creatorcontrib><creatorcontrib>Schaab, Christoph</creatorcontrib><creatorcontrib>Tebbe, Andreas</creatorcontrib><creatorcontrib>Becker, Karl-Friedrich</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of proteome research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gündisch, Sibylle</au><au>Grundner-Culemann, Kathrin</au><au>Wolff, Claudia</au><au>Schott, Christina</au><au>Reischauer, Bilge</au><au>Machatti, Manuela</au><au>Groelz, Daniel</au><au>Schaab, Christoph</au><au>Tebbe, Andreas</au><au>Becker, Karl-Friedrich</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Delayed Times to Tissue Fixation Result in Unpredictable Global Phosphoproteome Changes</atitle><jtitle>Journal of proteome research</jtitle><addtitle>J. Proteome Res</addtitle><date>2013-10-04</date><risdate>2013</risdate><volume>12</volume><issue>10</issue><spage>4424</spage><epage>4434</epage><pages>4424-4434</pages><issn>1535-3893</issn><eissn>1535-3907</eissn><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.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>23984901</pmid><doi>10.1021/pr400451z</doi><tpages>11</tpages></addata></record> |
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| ispartof | Journal of proteome research, 2013-10, Vol.12 (10), p.4424-4434 |
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| 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 |
| title | Delayed Times to Tissue Fixation Result in Unpredictable Global Phosphoproteome Changes |
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