Loading…

Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach

The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconC...

Full description

Saved in:
Bibliographic Details
Published in:Molecular & cellular proteomics 2011-12, Vol.10 (12), p.M111.007633-M111.007633
Main Authors: Carroll, Kathleen M, Simpson, Deborah M, Eyers, Claire E, Knight, Christopher G, Brownridge, Philip, Dunn, Warwick B, Winder, Catherine L, Lanthaler, Karin, Pir, Pinar, Malys, Naglis, Kell, Douglas B, Oliver, Stephen G, Gaskell, Simon J, Beynon, Robert J
Format: Article
Language:English
Subjects:
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
cited_by
cites
container_end_page M111.007633
container_issue 12
container_start_page M111.007633
container_title Molecular & cellular proteomics
container_volume 10
creator Carroll, Kathleen M
Simpson, Deborah M
Eyers, Claire E
Knight, Christopher G
Brownridge, Philip
Dunn, Warwick B
Winder, Catherine L
Lanthaler, Karin
Pir, Pinar
Malys, Naglis
Kell, Douglas B
Oliver, Stephen G
Gaskell, Simon J
Beynon, Robert J
description The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.
doi_str_mv 10.1074/mcp.M111.007633
format article
fullrecord <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_3237070</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>1028075996</sourcerecordid><originalsourceid>FETCH-LOGICAL-p298t-3464dd196d63ac550045fc799b998eae5b99368ad38756d3a22b4aada841df4d3</originalsourceid><addsrcrecordid>eNp9kUtrGzEUhUVIqN206-yCdsnGjp6jURYBY9IHpJRCuh6uJY2toBnJM5qG-feZUtckm6zOgfvxceAidEHJkhIlbhqTlj8opUtCVMH5CZpTyeVCi1KcHrsqZuhj3z8RwghV8gOaMao5pZLOkVtt-hiG7PB-gDb72hvIPrY41jjvHN6G0cQwZm9wgrx7hhH7Fo8O-nyLrUshjo1r818csIlNCm5y_TKxXa8eMaTURTC7T-ishtC7z4c8R7-_3D-uvy0efn79vl49LBLTZV5wUQhrqS5swcFISYiQtVFab7QuHTg5JS9KsLxUsrAcGNsIAAuloLYWlp-ju3_eNGwaZ820rINQpc430I1VBF-9vbR-V23jn4ozrogik-DqIOjifnB9rhrfGxcCtC4OfaWJVpIpRify-l2SElYSJbUuJvTy9arjnP9f4C87_ovv</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1028075996</pqid></control><display><type>article</type><title>Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach</title><source>Open Access: PubMed Central</source><source>ScienceDirect Journals</source><creator>Carroll, Kathleen M ; Simpson, Deborah M ; Eyers, Claire E ; Knight, Christopher G ; Brownridge, Philip ; Dunn, Warwick B ; Winder, Catherine L ; Lanthaler, Karin ; Pir, Pinar ; Malys, Naglis ; Kell, Douglas B ; Oliver, Stephen G ; Gaskell, Simon J ; Beynon, Robert J</creator><creatorcontrib>Carroll, Kathleen M ; Simpson, Deborah M ; Eyers, Claire E ; Knight, Christopher G ; Brownridge, Philip ; Dunn, Warwick B ; Winder, Catherine L ; Lanthaler, Karin ; Pir, Pinar ; Malys, Naglis ; Kell, Douglas B ; Oliver, Stephen G ; Gaskell, Simon J ; Beynon, Robert J</creatorcontrib><description>The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.</description><identifier>ISSN: 1535-9476</identifier><identifier>EISSN: 1535-9484</identifier><identifier>DOI: 10.1074/mcp.M111.007633</identifier><identifier>PMID: 21931151</identifier><language>eng</language><publisher>United States: The American Society for Biochemistry and Molecular Biology</publisher><subject>Amino Acid Sequence ; Concatamers ; Data processing ; DNA ; Enzymes ; Gene Expression ; Glycolysis ; Isoenzymes ; Isoenzymes - chemistry ; Isoenzymes - genetics ; Isoenzymes - metabolism ; Isotopes ; Mass spectroscopy ; Molecular Sequence Data ; Peptide Fragments - chemistry ; Peptide Fragments - standards ; Protein Processing, Post-Translational ; Proteolysis ; Proteomics ; Reference Standards ; Reproducibility of Results ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - enzymology ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae Proteins - chemistry ; Saccharomyces cerevisiae Proteins - genetics ; Saccharomyces cerevisiae Proteins - metabolism ; Tandem Mass Spectrometry - standards ; Tryptic peptides</subject><ispartof>Molecular &amp; cellular proteomics, 2011-12, Vol.10 (12), p.M111.007633-M111.007633</ispartof><rights>2011 by The American Society for Biochemistry and Molecular Biology, Inc. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237070/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237070/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,724,777,781,882,27905,27906,53772,53774</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21931151$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Carroll, Kathleen M</creatorcontrib><creatorcontrib>Simpson, Deborah M</creatorcontrib><creatorcontrib>Eyers, Claire E</creatorcontrib><creatorcontrib>Knight, Christopher G</creatorcontrib><creatorcontrib>Brownridge, Philip</creatorcontrib><creatorcontrib>Dunn, Warwick B</creatorcontrib><creatorcontrib>Winder, Catherine L</creatorcontrib><creatorcontrib>Lanthaler, Karin</creatorcontrib><creatorcontrib>Pir, Pinar</creatorcontrib><creatorcontrib>Malys, Naglis</creatorcontrib><creatorcontrib>Kell, Douglas B</creatorcontrib><creatorcontrib>Oliver, Stephen G</creatorcontrib><creatorcontrib>Gaskell, Simon J</creatorcontrib><creatorcontrib>Beynon, Robert J</creatorcontrib><title>Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach</title><title>Molecular &amp; cellular proteomics</title><addtitle>Mol Cell Proteomics</addtitle><description>The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.</description><subject>Amino Acid Sequence</subject><subject>Concatamers</subject><subject>Data processing</subject><subject>DNA</subject><subject>Enzymes</subject><subject>Gene Expression</subject><subject>Glycolysis</subject><subject>Isoenzymes</subject><subject>Isoenzymes - chemistry</subject><subject>Isoenzymes - genetics</subject><subject>Isoenzymes - metabolism</subject><subject>Isotopes</subject><subject>Mass spectroscopy</subject><subject>Molecular Sequence Data</subject><subject>Peptide Fragments - chemistry</subject><subject>Peptide Fragments - standards</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteolysis</subject><subject>Proteomics</subject><subject>Reference Standards</subject><subject>Reproducibility of Results</subject><subject>Saccharomyces cerevisiae</subject><subject>Saccharomyces cerevisiae - enzymology</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae Proteins - chemistry</subject><subject>Saccharomyces cerevisiae Proteins - genetics</subject><subject>Saccharomyces cerevisiae Proteins - metabolism</subject><subject>Tandem Mass Spectrometry - standards</subject><subject>Tryptic peptides</subject><issn>1535-9476</issn><issn>1535-9484</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><recordid>eNp9kUtrGzEUhUVIqN206-yCdsnGjp6jURYBY9IHpJRCuh6uJY2toBnJM5qG-feZUtckm6zOgfvxceAidEHJkhIlbhqTlj8opUtCVMH5CZpTyeVCi1KcHrsqZuhj3z8RwghV8gOaMao5pZLOkVtt-hiG7PB-gDb72hvIPrY41jjvHN6G0cQwZm9wgrx7hhH7Fo8O-nyLrUshjo1r818csIlNCm5y_TKxXa8eMaTURTC7T-ishtC7z4c8R7-_3D-uvy0efn79vl49LBLTZV5wUQhrqS5swcFISYiQtVFab7QuHTg5JS9KsLxUsrAcGNsIAAuloLYWlp-ju3_eNGwaZ820rINQpc430I1VBF-9vbR-V23jn4ozrogik-DqIOjifnB9rhrfGxcCtC4OfaWJVpIpRify-l2SElYSJbUuJvTy9arjnP9f4C87_ovv</recordid><startdate>20111201</startdate><enddate>20111201</enddate><creator>Carroll, Kathleen M</creator><creator>Simpson, Deborah M</creator><creator>Eyers, Claire E</creator><creator>Knight, Christopher G</creator><creator>Brownridge, Philip</creator><creator>Dunn, Warwick B</creator><creator>Winder, Catherine L</creator><creator>Lanthaler, Karin</creator><creator>Pir, Pinar</creator><creator>Malys, Naglis</creator><creator>Kell, Douglas B</creator><creator>Oliver, Stephen G</creator><creator>Gaskell, Simon J</creator><creator>Beynon, Robert J</creator><general>The American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>M7N</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20111201</creationdate><title>Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach</title><author>Carroll, Kathleen M ; Simpson, Deborah M ; Eyers, Claire E ; Knight, Christopher G ; Brownridge, Philip ; Dunn, Warwick B ; Winder, Catherine L ; Lanthaler, Karin ; Pir, Pinar ; Malys, Naglis ; Kell, Douglas B ; Oliver, Stephen G ; Gaskell, Simon J ; Beynon, Robert J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p298t-3464dd196d63ac550045fc799b998eae5b99368ad38756d3a22b4aada841df4d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Amino Acid Sequence</topic><topic>Concatamers</topic><topic>Data processing</topic><topic>DNA</topic><topic>Enzymes</topic><topic>Gene Expression</topic><topic>Glycolysis</topic><topic>Isoenzymes</topic><topic>Isoenzymes - chemistry</topic><topic>Isoenzymes - genetics</topic><topic>Isoenzymes - metabolism</topic><topic>Isotopes</topic><topic>Mass spectroscopy</topic><topic>Molecular Sequence Data</topic><topic>Peptide Fragments - chemistry</topic><topic>Peptide Fragments - standards</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteolysis</topic><topic>Proteomics</topic><topic>Reference Standards</topic><topic>Reproducibility of Results</topic><topic>Saccharomyces cerevisiae</topic><topic>Saccharomyces cerevisiae - enzymology</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae Proteins - chemistry</topic><topic>Saccharomyces cerevisiae Proteins - genetics</topic><topic>Saccharomyces cerevisiae Proteins - metabolism</topic><topic>Tandem Mass Spectrometry - standards</topic><topic>Tryptic peptides</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Carroll, Kathleen M</creatorcontrib><creatorcontrib>Simpson, Deborah M</creatorcontrib><creatorcontrib>Eyers, Claire E</creatorcontrib><creatorcontrib>Knight, Christopher G</creatorcontrib><creatorcontrib>Brownridge, Philip</creatorcontrib><creatorcontrib>Dunn, Warwick B</creatorcontrib><creatorcontrib>Winder, Catherine L</creatorcontrib><creatorcontrib>Lanthaler, Karin</creatorcontrib><creatorcontrib>Pir, Pinar</creatorcontrib><creatorcontrib>Malys, Naglis</creatorcontrib><creatorcontrib>Kell, Douglas B</creatorcontrib><creatorcontrib>Oliver, Stephen G</creatorcontrib><creatorcontrib>Gaskell, Simon J</creatorcontrib><creatorcontrib>Beynon, Robert J</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular &amp; cellular proteomics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Carroll, Kathleen M</au><au>Simpson, Deborah M</au><au>Eyers, Claire E</au><au>Knight, Christopher G</au><au>Brownridge, Philip</au><au>Dunn, Warwick B</au><au>Winder, Catherine L</au><au>Lanthaler, Karin</au><au>Pir, Pinar</au><au>Malys, Naglis</au><au>Kell, Douglas B</au><au>Oliver, Stephen G</au><au>Gaskell, Simon J</au><au>Beynon, Robert J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach</atitle><jtitle>Molecular &amp; cellular proteomics</jtitle><addtitle>Mol Cell Proteomics</addtitle><date>2011-12-01</date><risdate>2011</risdate><volume>10</volume><issue>12</issue><spage>M111.007633</spage><epage>M111.007633</epage><pages>M111.007633-M111.007633</pages><issn>1535-9476</issn><eissn>1535-9484</eissn><abstract>The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.</abstract><cop>United States</cop><pub>The American Society for Biochemistry and Molecular Biology</pub><pmid>21931151</pmid><doi>10.1074/mcp.M111.007633</doi><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 1535-9476
ispartof Molecular & cellular proteomics, 2011-12, Vol.10 (12), p.M111.007633-M111.007633
issn 1535-9476
1535-9484
language eng
recordid cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_3237070
source Open Access: PubMed Central; ScienceDirect Journals
subjects Amino Acid Sequence
Concatamers
Data processing
DNA
Enzymes
Gene Expression
Glycolysis
Isoenzymes
Isoenzymes - chemistry
Isoenzymes - genetics
Isoenzymes - metabolism
Isotopes
Mass spectroscopy
Molecular Sequence Data
Peptide Fragments - chemistry
Peptide Fragments - standards
Protein Processing, Post-Translational
Proteolysis
Proteomics
Reference Standards
Reproducibility of Results
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae Proteins - chemistry
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Tandem Mass Spectrometry - standards
Tryptic peptides
title Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach
url http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-20T07%3A19%3A20IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Absolute%20quantification%20of%20the%20glycolytic%20pathway%20in%20yeast:%20deployment%20of%20a%20complete%20QconCAT%20approach&rft.jtitle=Molecular%20&%20cellular%20proteomics&rft.au=Carroll,%20Kathleen%20M&rft.date=2011-12-01&rft.volume=10&rft.issue=12&rft.spage=M111.007633&rft.epage=M111.007633&rft.pages=M111.007633-M111.007633&rft.issn=1535-9476&rft.eissn=1535-9484&rft_id=info:doi/10.1074/mcp.M111.007633&rft_dat=%3Cproquest_pubme%3E1028075996%3C/proquest_pubme%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-p298t-3464dd196d63ac550045fc799b998eae5b99368ad38756d3a22b4aada841df4d3%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=1028075996&rft_id=info:pmid/21931151&rfr_iscdi=true