Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms
Drug resistance is often associated with upregulation of membrane-associated drug-efflux systems, and thus global membrane proteomics methods are valuable tools in the search for novel components of drug resistance phenotypes. Herein we have compared the microsomal proteome from the lung cancer cell...
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| Veröffentlicht in: | Proteomics (Weinheim) 2008-08, Vol.8 (15), p.3008-3018 |
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| creator | Eriksson, Hanna Lengqvist, Johan Hedlund, Joel Uhlén, Kristina Orre, Lukas M Bjellqvist, Bengt Persson, Bengt Lehtiö, Janne Jakobsson, Per-Johan |
| description | Drug resistance is often associated with upregulation of membrane-associated drug-efflux systems, and thus global membrane proteomics methods are valuable tools in the search for novel components of drug resistance phenotypes. Herein we have compared the microsomal proteome from the lung cancer cell line H69 and its isogenic Doxorubicin-resistant subcell line H69AR. The method used includes microsome preparation, iTRAQ labeling followed by narrow range peptide IEF in an immobilized pH-gradient (IPG-IEF) and LC-MS/MS analysis. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide IPG-IEF separation is demonstrated by its ability to: (i) lowering the complexity of the sample by two-thirds while keeping high proteome coverage (96%), (ii) providing high separation efficiency, and (iii) allowing for peptide validation and possibly identifications of post-transcriptional modifications. After analyzing one-fifth of the IEF fractions (effective pH range of 4.0-4.5), a total of 3704 proteins were identified, among which 527 were predicted to be membrane proteins. One of the proteins found to be differentially expressed was Serca 2, a calcium pump located in the ER membrane that potentially could result in changes of apoptotic response toward Doxorubicin. |
| doi_str_mv | 10.1002/pmic.200800174 |
| format | Article |
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Herein we have compared the microsomal proteome from the lung cancer cell line H69 and its isogenic Doxorubicin-resistant subcell line H69AR. The method used includes microsome preparation, iTRAQ labeling followed by narrow range peptide IEF in an immobilized pH-gradient (IPG-IEF) and LC-MS/MS analysis. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide IPG-IEF separation is demonstrated by its ability to: (i) lowering the complexity of the sample by two-thirds while keeping high proteome coverage (96%), (ii) providing high separation efficiency, and (iii) allowing for peptide validation and possibly identifications of post-transcriptional modifications. After analyzing one-fifth of the IEF fractions (effective pH range of 4.0-4.5), a total of 3704 proteins were identified, among which 527 were predicted to be membrane proteins. One of the proteins found to be differentially expressed was Serca 2, a calcium pump located in the ER membrane that potentially could result in changes of apoptotic response toward Doxorubicin.</description><identifier>ISSN: 1615-9853</identifier><identifier>ISSN: 1615-9861</identifier><identifier>EISSN: 1615-9861</identifier><identifier>DOI: 10.1002/pmic.200800174</identifier><identifier>PMID: 18654985</identifier><language>eng</language><publisher>Weinheim: Wiley-VCH Verlag</publisher><subject>Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Blotting, Western ; Cell Line, Tumor ; Chromatography, Liquid ; Drug resistance ; Fundamental and applied biological sciences. Psychology ; Humans ; Isoelectric focusing ; Isoelectric Focusing - methods ; Lung cancer ; Lung Neoplasms - metabolism ; Lung Neoplasms - pathology ; Medical sciences ; MEDICIN ; MEDICINE ; Membrane proteins ; Membrane Proteins - analysis ; Membrane Proteins - chemistry ; Membrane Proteins - isolation & purification ; Microsomes - metabolism ; Miscellaneous ; Models, Theoretical ; Nanotechnology ; Pneumology ; Proteins ; Proteomics - methods ; Quantitative proteomics ; Reproducibility of Results ; Small Cell Lung Carcinoma - metabolism ; Small Cell Lung Carcinoma - pathology ; Tandem Mass Spectrometry ; Tumors of the respiratory system and mediastinum</subject><ispartof>Proteomics (Weinheim), 2008-08, Vol.8 (15), p.3008-3018</ispartof><rights>Copyright © 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim</rights><rights>2008 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5764-776e3387f8d0005731e4336f7721b29743f7595bb83d7376d057985ef58df86a3</citedby><cites>FETCH-LOGICAL-c5764-776e3387f8d0005731e4336f7721b29743f7595bb83d7376d057985ef58df86a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fpmic.200800174$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fpmic.200800174$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>230,314,776,780,881,1411,27901,27902,45550,45551</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20552452$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18654985$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-16263$$DView record from Swedish Publication Index$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:117502778$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>Eriksson, Hanna</creatorcontrib><creatorcontrib>Lengqvist, Johan</creatorcontrib><creatorcontrib>Hedlund, Joel</creatorcontrib><creatorcontrib>Uhlén, Kristina</creatorcontrib><creatorcontrib>Orre, Lukas M</creatorcontrib><creatorcontrib>Bjellqvist, Bengt</creatorcontrib><creatorcontrib>Persson, Bengt</creatorcontrib><creatorcontrib>Lehtiö, Janne</creatorcontrib><creatorcontrib>Jakobsson, Per-Johan</creatorcontrib><title>Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms</title><title>Proteomics (Weinheim)</title><addtitle>Proteomics</addtitle><description>Drug resistance is often associated with upregulation of membrane-associated drug-efflux systems, and thus global membrane proteomics methods are valuable tools in the search for novel components of drug resistance phenotypes. Herein we have compared the microsomal proteome from the lung cancer cell line H69 and its isogenic Doxorubicin-resistant subcell line H69AR. The method used includes microsome preparation, iTRAQ labeling followed by narrow range peptide IEF in an immobilized pH-gradient (IPG-IEF) and LC-MS/MS analysis. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide IPG-IEF separation is demonstrated by its ability to: (i) lowering the complexity of the sample by two-thirds while keeping high proteome coverage (96%), (ii) providing high separation efficiency, and (iii) allowing for peptide validation and possibly identifications of post-transcriptional modifications. After analyzing one-fifth of the IEF fractions (effective pH range of 4.0-4.5), a total of 3704 proteins were identified, among which 527 were predicted to be membrane proteins. One of the proteins found to be differentially expressed was Serca 2, a calcium pump located in the ER membrane that potentially could result in changes of apoptotic response toward Doxorubicin.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>Blotting, Western</subject><subject>Cell Line, Tumor</subject><subject>Chromatography, Liquid</subject><subject>Drug resistance</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Humans</subject><subject>Isoelectric focusing</subject><subject>Isoelectric Focusing - methods</subject><subject>Lung cancer</subject><subject>Lung Neoplasms - metabolism</subject><subject>Lung Neoplasms - pathology</subject><subject>Medical sciences</subject><subject>MEDICIN</subject><subject>MEDICINE</subject><subject>Membrane proteins</subject><subject>Membrane Proteins - analysis</subject><subject>Membrane Proteins - chemistry</subject><subject>Membrane Proteins - isolation & purification</subject><subject>Microsomes - metabolism</subject><subject>Miscellaneous</subject><subject>Models, Theoretical</subject><subject>Nanotechnology</subject><subject>Pneumology</subject><subject>Proteins</subject><subject>Proteomics - methods</subject><subject>Quantitative proteomics</subject><subject>Reproducibility of Results</subject><subject>Small Cell Lung Carcinoma - metabolism</subject><subject>Small Cell Lung Carcinoma - pathology</subject><subject>Tandem Mass Spectrometry</subject><subject>Tumors of the respiratory system and mediastinum</subject><issn>1615-9853</issn><issn>1615-9861</issn><issn>1615-9861</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkctu1DAUhiMEohfYsgRv6IoZfIntZFkNMK1UbqIFxMZyHHswTeJgJwzzFLwyJ5poYIPY2Ec-37n4_7PsEcFLgjF93rfeLCnGBcZE5neyYyIIX5SFIHcPMWdH2UlK3yakKOX97IgUgueQOM5-vR91N_hBD_6HRa1tq6g7i_oYBhugd0K675ud7zao0zGGLYL8BgDbD762yKdgG2uG6A1ywYxpIl2IKA1j7W1CwaHU6qZBxsLRjJA2ujM2omiTT8MUw1zzVXc-telBds_pJtmH832a3bx6eb26WFy9XV-uzq8WhkuRL6QUlrFCuqLGGHPJiM0ZE05KSipaypw5yUteVQWrJZOiBgb-ax0valcIzU6zxb5v2tp-rFQffavjTgXt1fx0C5FVXDDMMfDP_sm_8B_PVYgb1fhREUEFA_xsj4OQ30ebBtX6NCkA4oYxKVGyoihxDuByD5oYUorWHToTrCaH1eSwOjgMBY_nzmPV2voPPlsKwNMZ0MnoxoFfxqcDRzHnNOcUuHLPbX1jd_8Zq969vlz9vcSsHhhofx5qdbxVAuTm6tObtWLXa_6Z0gv1Bfgne97poPQmwj43HygmDOOSgm-c_QZ42ttf</recordid><startdate>20080801</startdate><enddate>20080801</enddate><creator>Eriksson, Hanna</creator><creator>Lengqvist, Johan</creator><creator>Hedlund, Joel</creator><creator>Uhlén, Kristina</creator><creator>Orre, Lukas M</creator><creator>Bjellqvist, Bengt</creator><creator>Persson, Bengt</creator><creator>Lehtiö, Janne</creator><creator>Jakobsson, Per-Johan</creator><general>Wiley-VCH Verlag</general><general>WILEY-VCH Verlag</general><general>WILEY‐VCH Verlag</general><general>Wiley-VCH</general><scope>FBQ</scope><scope>BSCLL</scope><scope>IQODW</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>7X8</scope><scope>ADTPV</scope><scope>AOWAS</scope><scope>DG8</scope></search><sort><creationdate>20080801</creationdate><title>Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms</title><author>Eriksson, Hanna ; Lengqvist, Johan ; Hedlund, Joel ; Uhlén, Kristina ; Orre, Lukas M ; Bjellqvist, Bengt ; Persson, Bengt ; Lehtiö, Janne ; Jakobsson, Per-Johan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5764-776e3387f8d0005731e4336f7721b29743f7595bb83d7376d057985ef58df86a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>Blotting, Western</topic><topic>Cell Line, Tumor</topic><topic>Chromatography, Liquid</topic><topic>Drug resistance</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Humans</topic><topic>Isoelectric focusing</topic><topic>Isoelectric Focusing - methods</topic><topic>Lung cancer</topic><topic>Lung Neoplasms - metabolism</topic><topic>Lung Neoplasms - pathology</topic><topic>Medical sciences</topic><topic>MEDICIN</topic><topic>MEDICINE</topic><topic>Membrane proteins</topic><topic>Membrane Proteins - analysis</topic><topic>Membrane Proteins - chemistry</topic><topic>Membrane Proteins - isolation & purification</topic><topic>Microsomes - metabolism</topic><topic>Miscellaneous</topic><topic>Models, Theoretical</topic><topic>Nanotechnology</topic><topic>Pneumology</topic><topic>Proteins</topic><topic>Proteomics - methods</topic><topic>Quantitative proteomics</topic><topic>Reproducibility of Results</topic><topic>Small Cell Lung Carcinoma - metabolism</topic><topic>Small Cell Lung Carcinoma - pathology</topic><topic>Tandem Mass Spectrometry</topic><topic>Tumors of the respiratory system and mediastinum</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Eriksson, Hanna</creatorcontrib><creatorcontrib>Lengqvist, Johan</creatorcontrib><creatorcontrib>Hedlund, Joel</creatorcontrib><creatorcontrib>Uhlén, Kristina</creatorcontrib><creatorcontrib>Orre, Lukas M</creatorcontrib><creatorcontrib>Bjellqvist, Bengt</creatorcontrib><creatorcontrib>Persson, Bengt</creatorcontrib><creatorcontrib>Lehtiö, Janne</creatorcontrib><creatorcontrib>Jakobsson, Per-Johan</creatorcontrib><collection>AGRIS</collection><collection>Istex</collection><collection>Pascal-Francis</collection><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><collection>SwePub</collection><collection>SwePub Articles</collection><collection>SWEPUB Linköpings universitet</collection><jtitle>Proteomics (Weinheim)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Eriksson, Hanna</au><au>Lengqvist, Johan</au><au>Hedlund, Joel</au><au>Uhlén, Kristina</au><au>Orre, Lukas M</au><au>Bjellqvist, Bengt</au><au>Persson, Bengt</au><au>Lehtiö, Janne</au><au>Jakobsson, Per-Johan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms</atitle><jtitle>Proteomics (Weinheim)</jtitle><addtitle>Proteomics</addtitle><date>2008-08-01</date><risdate>2008</risdate><volume>8</volume><issue>15</issue><spage>3008</spage><epage>3018</epage><pages>3008-3018</pages><issn>1615-9853</issn><issn>1615-9861</issn><eissn>1615-9861</eissn><abstract>Drug resistance is often associated with upregulation of membrane-associated drug-efflux systems, and thus global membrane proteomics methods are valuable tools in the search for novel components of drug resistance phenotypes. Herein we have compared the microsomal proteome from the lung cancer cell line H69 and its isogenic Doxorubicin-resistant subcell line H69AR. The method used includes microsome preparation, iTRAQ labeling followed by narrow range peptide IEF in an immobilized pH-gradient (IPG-IEF) and LC-MS/MS analysis. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide IPG-IEF separation is demonstrated by its ability to: (i) lowering the complexity of the sample by two-thirds while keeping high proteome coverage (96%), (ii) providing high separation efficiency, and (iii) allowing for peptide validation and possibly identifications of post-transcriptional modifications. After analyzing one-fifth of the IEF fractions (effective pH range of 4.0-4.5), a total of 3704 proteins were identified, among which 527 were predicted to be membrane proteins. One of the proteins found to be differentially expressed was Serca 2, a calcium pump located in the ER membrane that potentially could result in changes of apoptotic response toward Doxorubicin.</abstract><cop>Weinheim</cop><pub>Wiley-VCH Verlag</pub><pmid>18654985</pmid><doi>10.1002/pmic.200800174</doi><tpages>11</tpages></addata></record> |
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| ispartof | Proteomics (Weinheim), 2008-08, Vol.8 (15), p.3008-3018 |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Analytical, structural and metabolic biochemistry Biological and medical sciences Blotting, Western Cell Line, Tumor Chromatography, Liquid Drug resistance Fundamental and applied biological sciences. Psychology Humans Isoelectric focusing Isoelectric Focusing - methods Lung cancer Lung Neoplasms - metabolism Lung Neoplasms - pathology Medical sciences MEDICIN MEDICINE Membrane proteins Membrane Proteins - analysis Membrane Proteins - chemistry Membrane Proteins - isolation & purification Microsomes - metabolism Miscellaneous Models, Theoretical Nanotechnology Pneumology Proteins Proteomics - methods Quantitative proteomics Reproducibility of Results Small Cell Lung Carcinoma - metabolism Small Cell Lung Carcinoma - pathology Tandem Mass Spectrometry Tumors of the respiratory system and mediastinum |
| title | Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms |
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