Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells
Cleavable isotope‐coded affinity tag (cICAT) reagents were utilized to identify and quantitate protein expression differences in control and inorganic phosphate‐treated murine MC3T3‐E1 osteoblast cells. Proteins extracted from control and treated cells were labeled with the light and heavy isotopic...
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Veröffentlicht in: | Electrophoresis 2004-05, Vol.25 (9), p.1342-1352 |
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creator | Conrads, Kelly A. Yu, Li-Rong Lucas, David A. Zhou, Ming Chan, King C. Simpson, Kerri A. Schaefer, Carl F. Issaq, Haleem J. Veenstra, Timothy D. Beck Jr, George R. Conrads, Thomas P. |
description | Cleavable isotope‐coded affinity tag (cICAT) reagents were utilized to identify and quantitate protein expression differences in control and inorganic phosphate‐treated murine MC3T3‐E1 osteoblast cells. Proteins extracted from control and treated cells were labeled with the light and heavy isotopic versions of cICAT reagents, respectively. The cICAT‐labeled samples were combined, proteolytically digested, and the cICAT‐derivatized peptides isolated using immobilized avidin chromatography. The cICAT‐labeled peptides were resolved into 96 fractions by strong cation‐exchange (SCX) liquid chromatography (LC). Analysis of the SCX‐LC cICAT peptide fractions by microcapillary reversed‐phase LC‐tandem mass spectrometry resulted in the identification and quantitation of 7227 unique peptides corresponding to 2501 proteins, or roughly 9% of the proteins currently predicted to be encoded by the mouse genome. A false positive analysis indicated a 98% confidence in the peptide identifications. To corroborate changes in abundance measured by cICAT with those detectable in traditionally prepared cell lysate, we chose to analyze cyclin D1. Cyclin D1 has been previously identified as a phosphate‐responsive gene and was likewise identified as a phosphate‐responsive protein in the current analysis. The 1.76‐fold increase in abundance in cyclin D1 determined from cICAT corresponds well with the 2.41‐fold increase as determined by Western blotting. These results demonstrate that quantitative proteomics is capable of providing a quantitative view of thousands of proteins in mammalian cells within a defined set of experiments. |
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Proteins extracted from control and treated cells were labeled with the light and heavy isotopic versions of cICAT reagents, respectively. The cICAT‐labeled samples were combined, proteolytically digested, and the cICAT‐derivatized peptides isolated using immobilized avidin chromatography. The cICAT‐labeled peptides were resolved into 96 fractions by strong cation‐exchange (SCX) liquid chromatography (LC). Analysis of the SCX‐LC cICAT peptide fractions by microcapillary reversed‐phase LC‐tandem mass spectrometry resulted in the identification and quantitation of 7227 unique peptides corresponding to 2501 proteins, or roughly 9% of the proteins currently predicted to be encoded by the mouse genome. A false positive analysis indicated a 98% confidence in the peptide identifications. To corroborate changes in abundance measured by cICAT with those detectable in traditionally prepared cell lysate, we chose to analyze cyclin D1. Cyclin D1 has been previously identified as a phosphate‐responsive gene and was likewise identified as a phosphate‐responsive protein in the current analysis. The 1.76‐fold increase in abundance in cyclin D1 determined from cICAT corresponds well with the 2.41‐fold increase as determined by Western blotting. These results demonstrate that quantitative proteomics is capable of providing a quantitative view of thousands of proteins in mammalian cells within a defined set of experiments.</description><identifier>ISSN: 0173-0835</identifier><identifier>EISSN: 1522-2683</identifier><identifier>DOI: 10.1002/elps.200405892</identifier><identifier>PMID: 15174057</identifier><language>eng</language><publisher>Weinheim: WILEY-VCH Verlag</publisher><subject>Amino Acid Sequence ; Animals ; Avidin - chemistry ; Cells, Cultured ; Chromatography, Liquid ; Cleavable isotope-coded affinity tag ; Gene Expression Regulation - drug effects ; Inorganic phosphate ; Isotopes - chemistry ; MC3T3-E1 cells ; Mice ; Molecular Sequence Data ; Osteoblast ; Osteoblasts - metabolism ; Phosphates - pharmacology ; Proteome ; Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><ispartof>Electrophoresis, 2004-05, Vol.25 (9), p.1342-1352</ispartof><rights>Copyright © 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4102-724769265c8e3ed4cf81db2df680996688f90f60937d16dbc76554d154c40c323</citedby><cites>FETCH-LOGICAL-c4102-724769265c8e3ed4cf81db2df680996688f90f60937d16dbc76554d154c40c323</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Felps.200405892$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1417,27924,27925,45575</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15174057$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Conrads, Kelly A.</creatorcontrib><creatorcontrib>Yu, Li-Rong</creatorcontrib><creatorcontrib>Lucas, David A.</creatorcontrib><creatorcontrib>Zhou, Ming</creatorcontrib><creatorcontrib>Chan, King C.</creatorcontrib><creatorcontrib>Simpson, Kerri A.</creatorcontrib><creatorcontrib>Schaefer, Carl F.</creatorcontrib><creatorcontrib>Issaq, Haleem J.</creatorcontrib><creatorcontrib>Veenstra, Timothy D.</creatorcontrib><creatorcontrib>Beck Jr, George R.</creatorcontrib><creatorcontrib>Conrads, Thomas P.</creatorcontrib><title>Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells</title><title>Electrophoresis</title><addtitle>ELECTROPHORESIS</addtitle><description>Cleavable isotope‐coded affinity tag (cICAT) reagents were utilized to identify and quantitate protein expression differences in control and inorganic phosphate‐treated murine MC3T3‐E1 osteoblast cells. Proteins extracted from control and treated cells were labeled with the light and heavy isotopic versions of cICAT reagents, respectively. The cICAT‐labeled samples were combined, proteolytically digested, and the cICAT‐derivatized peptides isolated using immobilized avidin chromatography. The cICAT‐labeled peptides were resolved into 96 fractions by strong cation‐exchange (SCX) liquid chromatography (LC). Analysis of the SCX‐LC cICAT peptide fractions by microcapillary reversed‐phase LC‐tandem mass spectrometry resulted in the identification and quantitation of 7227 unique peptides corresponding to 2501 proteins, or roughly 9% of the proteins currently predicted to be encoded by the mouse genome. A false positive analysis indicated a 98% confidence in the peptide identifications. To corroborate changes in abundance measured by cICAT with those detectable in traditionally prepared cell lysate, we chose to analyze cyclin D1. Cyclin D1 has been previously identified as a phosphate‐responsive gene and was likewise identified as a phosphate‐responsive protein in the current analysis. The 1.76‐fold increase in abundance in cyclin D1 determined from cICAT corresponds well with the 2.41‐fold increase as determined by Western blotting. These results demonstrate that quantitative proteomics is capable of providing a quantitative view of thousands of proteins in mammalian cells within a defined set of experiments.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Avidin - chemistry</subject><subject>Cells, Cultured</subject><subject>Chromatography, Liquid</subject><subject>Cleavable isotope-coded affinity tag</subject><subject>Gene Expression Regulation - drug effects</subject><subject>Inorganic phosphate</subject><subject>Isotopes - chemistry</subject><subject>MC3T3-E1 cells</subject><subject>Mice</subject><subject>Molecular Sequence Data</subject><subject>Osteoblast</subject><subject>Osteoblasts - metabolism</subject><subject>Phosphates - pharmacology</subject><subject>Proteome</subject><subject>Proteomics</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><issn>0173-0835</issn><issn>1522-2683</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkEtPGzEURq2KqgToliWaFbsJ1-_xEoUQkAJ9QFWpG8uxPcVlXoxnaPPvcZQIumNlyTrfkX0QOsYwxQDkzFddnBIABrxQ5AOaYE5ITkRB99AEsKQ5FJTvo4MY_0DCFGOf0D7mWKaFnCD9bTTNEAYzhGefdX07-LYONjONqdYxxKwts9C0_W_TpNvuoY3dgxl8Hho3Wu-yeuxD47ObGb2n-RxnbUyCVWXikFlfVfEIfSxNFf3n3XmIflzO72dX-fLL4np2vswtw0BySZgUighuC0-9Y7YssFsRV4oClBKiKEoFpQBFpcPCrawUnDOHObMMLCX0EJ1uvekLT6OPg65D3LzANL4do5Y4WRRn74IEmOCUQgKnW9D2bYy9L3XXh9r0a41Bb9rrTXv92j4NTnbmcVV794bvYidAbYG_ofLrd3R6vvx69788325DCvzvdWv6Ry0klVz_vF3oC0lvr-CX0N_pC6rpn28</recordid><startdate>20040501</startdate><enddate>20040501</enddate><creator>Conrads, Kelly A.</creator><creator>Yu, Li-Rong</creator><creator>Lucas, David A.</creator><creator>Zhou, Ming</creator><creator>Chan, King C.</creator><creator>Simpson, Kerri A.</creator><creator>Schaefer, Carl F.</creator><creator>Issaq, Haleem J.</creator><creator>Veenstra, Timothy D.</creator><creator>Beck Jr, George R.</creator><creator>Conrads, Thomas P.</creator><general>WILEY-VCH Verlag</general><general>WILEY‐VCH Verlag</general><scope>BSCLL</scope><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>7QO</scope><scope>7QP</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20040501</creationdate><title>Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells</title><author>Conrads, Kelly A. ; Yu, Li-Rong ; Lucas, David A. ; Zhou, Ming ; Chan, King C. ; Simpson, Kerri A. ; Schaefer, Carl F. ; Issaq, Haleem J. ; Veenstra, Timothy D. ; Beck Jr, George R. ; Conrads, Thomas P.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4102-724769265c8e3ed4cf81db2df680996688f90f60937d16dbc76554d154c40c323</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Avidin - chemistry</topic><topic>Cells, Cultured</topic><topic>Chromatography, Liquid</topic><topic>Cleavable isotope-coded affinity tag</topic><topic>Gene Expression Regulation - drug effects</topic><topic>Inorganic phosphate</topic><topic>Isotopes - chemistry</topic><topic>MC3T3-E1 cells</topic><topic>Mice</topic><topic>Molecular Sequence Data</topic><topic>Osteoblast</topic><topic>Osteoblasts - metabolism</topic><topic>Phosphates - pharmacology</topic><topic>Proteome</topic><topic>Proteomics</topic><topic>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Conrads, Kelly A.</creatorcontrib><creatorcontrib>Yu, Li-Rong</creatorcontrib><creatorcontrib>Lucas, David A.</creatorcontrib><creatorcontrib>Zhou, Ming</creatorcontrib><creatorcontrib>Chan, King C.</creatorcontrib><creatorcontrib>Simpson, Kerri A.</creatorcontrib><creatorcontrib>Schaefer, Carl F.</creatorcontrib><creatorcontrib>Issaq, Haleem J.</creatorcontrib><creatorcontrib>Veenstra, Timothy D.</creatorcontrib><creatorcontrib>Beck Jr, George R.</creatorcontrib><creatorcontrib>Conrads, Thomas P.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Electrophoresis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Conrads, Kelly A.</au><au>Yu, Li-Rong</au><au>Lucas, David A.</au><au>Zhou, Ming</au><au>Chan, King C.</au><au>Simpson, Kerri A.</au><au>Schaefer, Carl F.</au><au>Issaq, Haleem J.</au><au>Veenstra, Timothy D.</au><au>Beck Jr, George R.</au><au>Conrads, Thomas P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells</atitle><jtitle>Electrophoresis</jtitle><addtitle>ELECTROPHORESIS</addtitle><date>2004-05-01</date><risdate>2004</risdate><volume>25</volume><issue>9</issue><spage>1342</spage><epage>1352</epage><pages>1342-1352</pages><issn>0173-0835</issn><eissn>1522-2683</eissn><abstract>Cleavable isotope‐coded affinity tag (cICAT) reagents were utilized to identify and quantitate protein expression differences in control and inorganic phosphate‐treated murine MC3T3‐E1 osteoblast cells. Proteins extracted from control and treated cells were labeled with the light and heavy isotopic versions of cICAT reagents, respectively. The cICAT‐labeled samples were combined, proteolytically digested, and the cICAT‐derivatized peptides isolated using immobilized avidin chromatography. The cICAT‐labeled peptides were resolved into 96 fractions by strong cation‐exchange (SCX) liquid chromatography (LC). Analysis of the SCX‐LC cICAT peptide fractions by microcapillary reversed‐phase LC‐tandem mass spectrometry resulted in the identification and quantitation of 7227 unique peptides corresponding to 2501 proteins, or roughly 9% of the proteins currently predicted to be encoded by the mouse genome. A false positive analysis indicated a 98% confidence in the peptide identifications. To corroborate changes in abundance measured by cICAT with those detectable in traditionally prepared cell lysate, we chose to analyze cyclin D1. Cyclin D1 has been previously identified as a phosphate‐responsive gene and was likewise identified as a phosphate‐responsive protein in the current analysis. The 1.76‐fold increase in abundance in cyclin D1 determined from cICAT corresponds well with the 2.41‐fold increase as determined by Western blotting. These results demonstrate that quantitative proteomics is capable of providing a quantitative view of thousands of proteins in mammalian cells within a defined set of experiments.</abstract><cop>Weinheim</cop><pub>WILEY-VCH Verlag</pub><pmid>15174057</pmid><doi>10.1002/elps.200405892</doi><tpages>11</tpages></addata></record> |
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subjects | Amino Acid Sequence Animals Avidin - chemistry Cells, Cultured Chromatography, Liquid Cleavable isotope-coded affinity tag Gene Expression Regulation - drug effects Inorganic phosphate Isotopes - chemistry MC3T3-E1 cells Mice Molecular Sequence Data Osteoblast Osteoblasts - metabolism Phosphates - pharmacology Proteome Proteomics Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization |
title | Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells |
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