Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma
Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MR...
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| Veröffentlicht in: | Journal of proteome research 2013-12, Vol.12 (12), p.5996-6003 |
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| creator | Lin, De Alborn, William E Slebos, Robbert J. C Liebler, Daniel C |
| description | Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978–0.995) between 10 and 640 ng/mL for the target proteins spiked into a “mock plasma” matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67–0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids. |
| doi_str_mv | 10.1021/pr400877e |
| format | Article |
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C ; Liebler, Daniel C</creator><creatorcontrib>Lin, De ; Alborn, William E ; Slebos, Robbert J. C ; Liebler, Daniel C</creatorcontrib><description>Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978–0.995) between 10 and 640 ng/mL for the target proteins spiked into a “mock plasma” matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67–0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.</description><identifier>ISSN: 1535-3893</identifier><identifier>ISSN: 1535-3907</identifier><identifier>EISSN: 1535-3907</identifier><identifier>DOI: 10.1021/pr400877e</identifier><identifier>PMID: 24224610</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acid Sequence ; Animals ; antibodies ; Antibodies - chemistry ; Antigens, CD - blood ; Antigens, CD - genetics ; biomarkers ; Biomarkers, Tumor - blood ; Biomarkers, Tumor - genetics ; bovine serum albumin ; Carcinoma - blood ; Carcinoma - diagnosis ; Carcinoma - genetics ; cartilage ; Cartilage Oligomeric Matrix Protein - blood ; Cartilage Oligomeric Matrix Protein - genetics ; Cattle ; Colonic Neoplasms - blood ; Colonic Neoplasms - diagnosis ; Colonic Neoplasms - genetics ; colorectal neoplasms ; detection limit ; Endoglin ; enzyme inhibitors ; enzyme-linked immunosorbent assay ; Enzyme-Linked Immunosorbent Assay - statistics & numerical data ; gelatinase B ; GPI-Linked Proteins - blood ; GPI-Linked Proteins - genetics ; Hernia - blood ; Hernia - diagnosis ; Hernia - genetics ; Humans ; Immunoprecipitation - statistics & numerical data ; isotope dilution technique ; mass spectrometry ; Mass Spectrometry - methods ; Matrix Metalloproteinase 9 - blood ; Matrix Metalloproteinase 9 - genetics ; Molecular Sequence Data ; monitoring ; patients ; polyacrylamide gel electrophoresis ; precipitin tests ; proteome ; quantitative analysis ; Receptors, Cell Surface - blood ; Receptors, Cell Surface - genetics ; stable isotopes ; Technical Note ; Thrombospondins - blood ; Thrombospondins - genetics ; Tissue Inhibitor of Metalloproteinase-1 - blood ; Tissue Inhibitor of Metalloproteinase-1 - genetics</subject><ispartof>Journal of proteome research, 2013-12, Vol.12 (12), p.5996-6003</ispartof><rights>Copyright © 2013 American Chemical Society</rights><rights>Copyright © 2013 American Chemical Society 2013 American Chemical Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a438t-c0c6ddafea4e8355e3fc132dbecfc7c4f1277a18c5138dd06b8f25147846e02b3</citedby><cites>FETCH-LOGICAL-a438t-c0c6ddafea4e8355e3fc132dbecfc7c4f1277a18c5138dd06b8f25147846e02b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/pr400877e$$EPDF$$P50$$Gacs$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/pr400877e$$EHTML$$P50$$Gacs$$Hfree_for_read</linktohtml><link.rule.ids>230,314,776,780,881,2752,27053,27901,27902,56713,56763</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24224610$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lin, De</creatorcontrib><creatorcontrib>Alborn, William E</creatorcontrib><creatorcontrib>Slebos, Robbert J. C</creatorcontrib><creatorcontrib>Liebler, Daniel C</creatorcontrib><title>Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma</title><title>Journal of proteome research</title><addtitle>J. Proteome Res</addtitle><description>Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978–0.995) between 10 and 640 ng/mL for the target proteins spiked into a “mock plasma” matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67–0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>antibodies</subject><subject>Antibodies - chemistry</subject><subject>Antigens, CD - blood</subject><subject>Antigens, CD - genetics</subject><subject>biomarkers</subject><subject>Biomarkers, Tumor - blood</subject><subject>Biomarkers, Tumor - genetics</subject><subject>bovine serum albumin</subject><subject>Carcinoma - blood</subject><subject>Carcinoma - diagnosis</subject><subject>Carcinoma - genetics</subject><subject>cartilage</subject><subject>Cartilage Oligomeric Matrix Protein - blood</subject><subject>Cartilage Oligomeric Matrix Protein - genetics</subject><subject>Cattle</subject><subject>Colonic Neoplasms - blood</subject><subject>Colonic Neoplasms - diagnosis</subject><subject>Colonic Neoplasms - genetics</subject><subject>colorectal neoplasms</subject><subject>detection limit</subject><subject>Endoglin</subject><subject>enzyme inhibitors</subject><subject>enzyme-linked immunosorbent assay</subject><subject>Enzyme-Linked Immunosorbent Assay - statistics & numerical data</subject><subject>gelatinase B</subject><subject>GPI-Linked Proteins - blood</subject><subject>GPI-Linked Proteins - genetics</subject><subject>Hernia - blood</subject><subject>Hernia - diagnosis</subject><subject>Hernia - genetics</subject><subject>Humans</subject><subject>Immunoprecipitation - statistics & numerical data</subject><subject>isotope dilution technique</subject><subject>mass spectrometry</subject><subject>Mass Spectrometry - methods</subject><subject>Matrix Metalloproteinase 9 - blood</subject><subject>Matrix Metalloproteinase 9 - genetics</subject><subject>Molecular Sequence Data</subject><subject>monitoring</subject><subject>patients</subject><subject>polyacrylamide gel electrophoresis</subject><subject>precipitin tests</subject><subject>proteome</subject><subject>quantitative analysis</subject><subject>Receptors, Cell Surface - blood</subject><subject>Receptors, Cell Surface - genetics</subject><subject>stable isotopes</subject><subject>Technical Note</subject><subject>Thrombospondins - blood</subject><subject>Thrombospondins - genetics</subject><subject>Tissue Inhibitor of Metalloproteinase-1 - blood</subject><subject>Tissue Inhibitor of Metalloproteinase-1 - genetics</subject><issn>1535-3893</issn><issn>1535-3907</issn><issn>1535-3907</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>N~.</sourceid><sourceid>EIF</sourceid><recordid>eNqFkV1rFDEUhoMotlYv_AOSG0EvRvM1SXojrEttF7ZY_LgO2cxJmzqTjElG6YX_3Vm2XSoUvErIeXhyznkReknJO0oYfT9mQYhWCh6hQ9rytuHHRD2-u-tjfoCelXJNCG0V4U_RAROMCUnJIfqzTMNocygp4uTxRU4VQsSrYZhiGjO4MIZqa0ixOZ_6GsYe8BewbvuCz1MMNeUQL_HvUK_wyXr1dYF9ynhRir3ZCj-GNNj8AzJe2tiFzlYoeP7gordlsM_RE2_7Ai9uzyP0_dPJt-VZs_58ulou1o0VXNfGESe7znqwAjRvW-DeUc66DTjvlBOeMqUs1a6lXHcdkRvtWUuF0kICYRt-hD7svOO0GaBzEGu2vRlzmJu7MckG828lhitzmX4ZrqVgUsyCN7eCnH5OUKoZQnHQ9zZCmophpJ33TDQh_0WpkJIrydkWfbtDXU6lZPD7jigx22TNPtmZfXV_hD15F-UMvN4B1hVznaYc540-IPoL6ZCtUA</recordid><startdate>20131206</startdate><enddate>20131206</enddate><creator>Lin, De</creator><creator>Alborn, William E</creator><creator>Slebos, Robbert J. C</creator><creator>Liebler, Daniel C</creator><general>American Chemical Society</general><scope>N~.</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>7S9</scope><scope>L.6</scope><scope>5PM</scope></search><sort><creationdate>20131206</creationdate><title>Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma</title><author>Lin, De ; Alborn, William E ; Slebos, Robbert J. C ; Liebler, Daniel C</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a438t-c0c6ddafea4e8355e3fc132dbecfc7c4f1277a18c5138dd06b8f25147846e02b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>antibodies</topic><topic>Antibodies - chemistry</topic><topic>Antigens, CD - blood</topic><topic>Antigens, CD - genetics</topic><topic>biomarkers</topic><topic>Biomarkers, Tumor - blood</topic><topic>Biomarkers, Tumor - genetics</topic><topic>bovine serum albumin</topic><topic>Carcinoma - blood</topic><topic>Carcinoma - diagnosis</topic><topic>Carcinoma - genetics</topic><topic>cartilage</topic><topic>Cartilage Oligomeric Matrix Protein - blood</topic><topic>Cartilage Oligomeric Matrix Protein - genetics</topic><topic>Cattle</topic><topic>Colonic Neoplasms - blood</topic><topic>Colonic Neoplasms - diagnosis</topic><topic>Colonic Neoplasms - genetics</topic><topic>colorectal neoplasms</topic><topic>detection limit</topic><topic>Endoglin</topic><topic>enzyme inhibitors</topic><topic>enzyme-linked immunosorbent assay</topic><topic>Enzyme-Linked Immunosorbent Assay - statistics & numerical data</topic><topic>gelatinase B</topic><topic>GPI-Linked Proteins - blood</topic><topic>GPI-Linked Proteins - genetics</topic><topic>Hernia - blood</topic><topic>Hernia - diagnosis</topic><topic>Hernia - genetics</topic><topic>Humans</topic><topic>Immunoprecipitation - statistics & numerical data</topic><topic>isotope dilution technique</topic><topic>mass spectrometry</topic><topic>Mass Spectrometry - methods</topic><topic>Matrix Metalloproteinase 9 - blood</topic><topic>Matrix Metalloproteinase 9 - genetics</topic><topic>Molecular Sequence Data</topic><topic>monitoring</topic><topic>patients</topic><topic>polyacrylamide gel electrophoresis</topic><topic>precipitin tests</topic><topic>proteome</topic><topic>quantitative analysis</topic><topic>Receptors, Cell Surface - blood</topic><topic>Receptors, Cell Surface - genetics</topic><topic>stable isotopes</topic><topic>Technical Note</topic><topic>Thrombospondins - blood</topic><topic>Thrombospondins - genetics</topic><topic>Tissue Inhibitor of Metalloproteinase-1 - blood</topic><topic>Tissue Inhibitor of Metalloproteinase-1 - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lin, De</creatorcontrib><creatorcontrib>Alborn, William E</creatorcontrib><creatorcontrib>Slebos, Robbert J. C</creatorcontrib><creatorcontrib>Liebler, Daniel C</creatorcontrib><collection>American Chemical Society (ACS) Open Access</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>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of proteome research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lin, De</au><au>Alborn, William E</au><au>Slebos, Robbert J. C</au><au>Liebler, Daniel C</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma</atitle><jtitle>Journal of proteome research</jtitle><addtitle>J. Proteome Res</addtitle><date>2013-12-06</date><risdate>2013</risdate><volume>12</volume><issue>12</issue><spage>5996</spage><epage>6003</epage><pages>5996-6003</pages><issn>1535-3893</issn><issn>1535-3907</issn><eissn>1535-3907</eissn><abstract>Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978–0.995) between 10 and 640 ng/mL for the target proteins spiked into a “mock plasma” matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67–0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>24224610</pmid><doi>10.1021/pr400877e</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Sequence Animals antibodies Antibodies - chemistry Antigens, CD - blood Antigens, CD - genetics biomarkers Biomarkers, Tumor - blood Biomarkers, Tumor - genetics bovine serum albumin Carcinoma - blood Carcinoma - diagnosis Carcinoma - genetics cartilage Cartilage Oligomeric Matrix Protein - blood Cartilage Oligomeric Matrix Protein - genetics Cattle Colonic Neoplasms - blood Colonic Neoplasms - diagnosis Colonic Neoplasms - genetics colorectal neoplasms detection limit Endoglin enzyme inhibitors enzyme-linked immunosorbent assay Enzyme-Linked Immunosorbent Assay - statistics & numerical data gelatinase B GPI-Linked Proteins - blood GPI-Linked Proteins - genetics Hernia - blood Hernia - diagnosis Hernia - genetics Humans Immunoprecipitation - statistics & numerical data isotope dilution technique mass spectrometry Mass Spectrometry - methods Matrix Metalloproteinase 9 - blood Matrix Metalloproteinase 9 - genetics Molecular Sequence Data monitoring patients polyacrylamide gel electrophoresis precipitin tests proteome quantitative analysis Receptors, Cell Surface - blood Receptors, Cell Surface - genetics stable isotopes Technical Note Thrombospondins - blood Thrombospondins - genetics Tissue Inhibitor of Metalloproteinase-1 - blood Tissue Inhibitor of Metalloproteinase-1 - genetics |
| title | Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma |
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