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Article: Combinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications

TitleCombinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications
Authors
Issue Date2011
PublisherRoyal Society of Chemistry. The Journal's web site is located at http://www.rsc.org/is/journals/current/mbs/mbspub.htm
Citation
Molecular Biosystems, 2011, v. 7 n. 5, p. 1399-1408 How to Cite?
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.
Persistent Identifierhttp://hdl.handle.net/10722/147631
ISSN
2014 Impact Factor: 3.210
2014 SCImago Journal Rankings: 1.205
ISI Accession Number ID
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.

References

 

DC FieldValueLanguage
dc.contributor.authorLau, Een_HK
dc.contributor.authorLam, MPYen_HK
dc.contributor.authorSiu, SOen_HK
dc.contributor.authorKong, RPWen_HK
dc.contributor.authorChan, WLen_HK
dc.contributor.authorZhou, Zen_HK
dc.contributor.authorHuang, Jen_HK
dc.contributor.authorLo, Cen_HK
dc.contributor.authorChu, IKen_HK
dc.date.accessioned2012-05-29T06:05:06Z-
dc.date.available2012-05-29T06:05:06Z-
dc.date.issued2011en_HK
dc.identifier.citationMolecular Biosystems, 2011, v. 7 n. 5, p. 1399-1408en_HK
dc.identifier.issn1742-206Xen_HK
dc.identifier.urihttp://hdl.handle.net/10722/147631-
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.en_HK
dc.languageengen_US
dc.publisherRoyal Society of Chemistry. The Journal's web site is located at http://www.rsc.org/is/journals/current/mbs/mbspub.htmen_HK
dc.relation.ispartofMolecular BioSystemsen_HK
dc.subject.meshAmino Acid Sequenceen_US
dc.subject.meshArabidopsis Proteins - Analysisen_US
dc.subject.meshCationsen_US
dc.subject.meshChloroplasts - Metabolismen_US
dc.subject.meshChromatography, Ion Exchange - Methodsen_US
dc.subject.meshChromatography, Liquid - Methodsen_US
dc.subject.meshMass Spectrometry - Methodsen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshPeptides - Analysisen_US
dc.subject.meshProteome - Analysisen_US
dc.subject.meshProteomics - Methodsen_US
dc.subject.meshReproducibility Of Resultsen_US
dc.titleCombinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applicationsen_HK
dc.typeArticleen_HK
dc.identifier.emailZhou, Z: zhongjun@hkucc.hku.hken_HK
dc.identifier.emailLo, C: clivelo@hkucc.hku.hken_HK
dc.identifier.emailChu, IK: ivankchu@hku.hken_HK
dc.identifier.authorityZhou, Z=rp00503en_HK
dc.identifier.authorityLo, C=rp00751en_HK
dc.identifier.authorityChu, IK=rp00683en_HK
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1039/c1mb05010aen_HK
dc.identifier.pmid21350782en_HK
dc.identifier.scopuseid_2-s2.0-79954497043en_HK
dc.identifier.hkuros186825-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-79954497043&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume7en_HK
dc.identifier.issue5en_HK
dc.identifier.spage1399en_HK
dc.identifier.epage1408en_HK
dc.identifier.isiWOS:000289367200004-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridLau, E=35302963200en_HK
dc.identifier.scopusauthoridLam, MPY=35302594800en_HK
dc.identifier.scopusauthoridSiu, SO=8603087200en_HK
dc.identifier.scopusauthoridKong, RPW=35217869000en_HK
dc.identifier.scopusauthoridChan, WL=7403918042en_HK
dc.identifier.scopusauthoridZhou, Z=8631856300en_HK
dc.identifier.scopusauthoridHuang, J=37088635900en_HK
dc.identifier.scopusauthoridLo, C=15737175700en_HK
dc.identifier.scopusauthoridChu, IK=7103327484en_HK

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