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

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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
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
2011 Impact Factor: 3.534
2011 SCImago Journal Rankings: 0.522
DOIhttp://dx.doi.org/10.1039/c1mb05010a
ReferencesReferences in Scopus
DC Field
Value
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
2011 Impact Factor: 3.534
2011 SCImago Journal Rankings: 0.522
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
Author Affiliations
  1. The University of Hong Kong
  2. Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences Chinese Academy of Sciences