File Download
 
Links for fulltext
(May Require Subscription)
 
Supplementary

Article: Combinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications
  • Basic View
  • Metadata View
  • XML View
TitleCombinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications
 
AuthorsLau, E1
Lam, MPY1
Siu, SO1
Kong, RPW1
Chan, WL1 1
Zhou, Z1
Huang, J2
Lo, C1
Chu, IK1
 
Issue Date2011
 
PublisherRoyal Society of Chemistry. The Journal's web site is located at http://www.rsc.org/is/journals/current/mbs/mbspub.htm
 
CitationMolecular Biosystems, 2011, v. 7 n. 5, p. 1399-1408 [How to Cite?]
DOI: http://dx.doi.org/10.1039/c1mb05010a
 
AbstractExtensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility. © The Royal Society of Chemistry 2011.
 
ISSN1742-206X
2012 Impact Factor: 3.35
2012 SCImago Journal Rankings: 1.388
 
DOIhttp://dx.doi.org/10.1039/c1mb05010a
 
ISI Accession Number IDWOS:000289367200004
Funding AgencyGrant Number
Hong Kong Research Grants CouncilHKU7018/09P
HKU3/07C
HKU7733/10M
Hong Kong Special Administrative Region, China
Funding Information:

This study was supported by the Hong Kong Research Grants Council (project nos. HKU7018/09P, HKU3/07C and HKU7733/10M), Hong Kong Special Administrative Region, China. E.L. and M.P.Y.L. thank the Hong Kong RGC for supporting their studentships.

 
ReferencesReferences in Scopus
 
DC FieldValue
dc.contributor.authorLau, E
 
dc.contributor.authorLam, MPY
 
dc.contributor.authorSiu, SO
 
dc.contributor.authorKong, RPW
 
dc.contributor.authorChan, WL
 
dc.contributor.authorZhou, Z
 
dc.contributor.authorHuang, J
 
dc.contributor.authorLo, C
 
dc.contributor.authorChu, IK
 
dc.date.accessioned2012-05-29T06:05:06Z
 
dc.date.available2012-05-29T06:05:06Z
 
dc.date.issued2011
 
dc.description.abstractExtensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility. © The Royal Society of Chemistry 2011.
 
dc.description.natureLink_to_subscribed_fulltext
 
dc.identifier.citationMolecular Biosystems, 2011, v. 7 n. 5, p. 1399-1408 [How to Cite?]
DOI: http://dx.doi.org/10.1039/c1mb05010a
 
dc.identifier.doihttp://dx.doi.org/10.1039/c1mb05010a
 
dc.identifier.epage1408
 
dc.identifier.hkuros186825
 
dc.identifier.isiWOS:000289367200004
Funding AgencyGrant Number
Hong Kong Research Grants CouncilHKU7018/09P
HKU3/07C
HKU7733/10M
Hong Kong Special Administrative Region, China
Funding Information:

This study was supported by the Hong Kong Research Grants Council (project nos. HKU7018/09P, HKU3/07C and HKU7733/10M), Hong Kong Special Administrative Region, China. E.L. and M.P.Y.L. thank the Hong Kong RGC for supporting their studentships.

 
dc.identifier.issn1742-206X
2012 Impact Factor: 3.35
2012 SCImago Journal Rankings: 1.388
 
dc.identifier.issue5
 
dc.identifier.pmid21350782
 
dc.identifier.scopuseid_2-s2.0-79954497043
 
dc.identifier.spage1399
 
dc.identifier.urihttp://hdl.handle.net/10722/147631
 
dc.identifier.volume7
 
dc.languageeng
 
dc.publisherRoyal Society of Chemistry. The Journal's web site is located at http://www.rsc.org/is/journals/current/mbs/mbspub.htm
 
dc.publisher.placeUnited Kingdom
 
dc.relation.ispartofMolecular BioSystems
 
dc.relation.referencesReferences in Scopus
 
dc.subject.meshAmino Acid Sequence
 
dc.subject.meshArabidopsis Proteins - Analysis
 
dc.subject.meshCations
 
dc.subject.meshChloroplasts - Metabolism
 
dc.subject.meshChromatography, Ion Exchange - Methods
 
dc.subject.meshChromatography, Liquid - Methods
 
dc.subject.meshMass Spectrometry - Methods
 
dc.subject.meshMolecular Sequence Data
 
dc.subject.meshPeptides - Analysis
 
dc.subject.meshProteome - Analysis
 
dc.subject.meshProteomics - Methods
 
dc.subject.meshReproducibility Of Results
 
dc.titleCombinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications
 
dc.typeArticle
 
<?xml encoding="utf-8" version="1.0"?>
<item><contributor.author>Lau, E</contributor.author>
<contributor.author>Lam, MPY</contributor.author>
<contributor.author>Siu, SO</contributor.author>
<contributor.author>Kong, RPW</contributor.author>
<contributor.author>Chan, WL</contributor.author>
<contributor.author>Zhou, Z</contributor.author>
<contributor.author>Huang, J</contributor.author>
<contributor.author>Lo, C</contributor.author>
<contributor.author>Chu, IK</contributor.author>
<date.accessioned>2012-05-29T06:05:06Z</date.accessioned>
<date.available>2012-05-29T06:05:06Z</date.available>
<date.issued>2011</date.issued>
<identifier.citation>Molecular Biosystems, 2011, v. 7 n. 5, p. 1399-1408</identifier.citation>
<identifier.issn>1742-206X</identifier.issn>
<identifier.uri>http://hdl.handle.net/10722/147631</identifier.uri>
<description.abstract>Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility. &#169; The Royal Society of Chemistry 2011.</description.abstract>
<language>eng</language>
<publisher>Royal Society of Chemistry. The Journal&apos;s web site is located at http://www.rsc.org/is/journals/current/mbs/mbspub.htm</publisher>
<relation.ispartof>Molecular BioSystems</relation.ispartof>
<subject.mesh>Amino Acid Sequence</subject.mesh>
<subject.mesh>Arabidopsis Proteins - Analysis</subject.mesh>
<subject.mesh>Cations</subject.mesh>
<subject.mesh>Chloroplasts - Metabolism</subject.mesh>
<subject.mesh>Chromatography, Ion Exchange - Methods</subject.mesh>
<subject.mesh>Chromatography, Liquid - Methods</subject.mesh>
<subject.mesh>Mass Spectrometry - Methods</subject.mesh>
<subject.mesh>Molecular Sequence Data</subject.mesh>
<subject.mesh>Peptides - Analysis</subject.mesh>
<subject.mesh>Proteome - Analysis</subject.mesh>
<subject.mesh>Proteomics - Methods</subject.mesh>
<subject.mesh>Reproducibility Of Results</subject.mesh>
<title>Combinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications</title>
<type>Article</type>
<description.nature>Link_to_subscribed_fulltext</description.nature>
<identifier.doi>10.1039/c1mb05010a</identifier.doi>
<identifier.pmid>21350782</identifier.pmid>
<identifier.scopus>eid_2-s2.0-79954497043</identifier.scopus>
<identifier.hkuros>186825</identifier.hkuros>
<relation.references>http://www.scopus.com/mlt/select.url?eid=2-s2.0-79954497043&amp;selection=ref&amp;src=s&amp;origin=recordpage</relation.references>
<identifier.volume>7</identifier.volume>
<identifier.issue>5</identifier.issue>
<identifier.spage>1399</identifier.spage>
<identifier.epage>1408</identifier.epage>
<identifier.isi>WOS:000289367200004</identifier.isi>
<publisher.place>United Kingdom</publisher.place>
</item>
Author Affiliations
  1. The University of Hong Kong
  2. Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences Chinese Academy of Sciences