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Article: Development of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approach

TitleDevelopment of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approach
Authors
KeywordsHigh-/low-pH RP-RP
Liquid chromatography
MDLC
Proteomics
RP-SCX-RP
Two-dimensional
Issue Date2011
PublisherElsevier BV. The Journal's web site is located at http://www.elsevier.com/locate/chroma
Citation
Journal Of Chromatography A, 2011, v. 1218 n. 23, p. 3681-3688 How to Cite?
AbstractPreviously, we described an online high-/low-pH RP-RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP-RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP-RP separation. Relative to the RP-RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample. © 2011 Elsevier B.V.
Persistent Identifierhttp://hdl.handle.net/10722/135040
ISSN
2015 Impact Factor: 3.926
2015 SCImago Journal Rankings: 1.774
ISI Accession Number ID
Funding AgencyGrant Number
Hong Kong Research Grants CouncilHKU7018/09P
HKU7733/10M
Hong Kong Special Administrative Region, China
Science and Technology Development Fund of Macau045/2007
058/2009
Research Committee, University of MacauUL017
Funding Information:

This study was supported by the Hong Kong Research Grants Council (project nos. HKU7018/09P and HKU7733/10M), Hong Kong Special Administrative Region, China, and by grants from the Science and Technology Development Fund of Macau (Ref. No. 045/2007 and 058/2009) and the Research Committee, University of Macau (Ref. No. UL017). Ricky P.W. Kong thanks the Hong Kong RGC for supporting his studentship. The authors would like to thank Dr. Ronald Pang for providing the mouse embryonic mouse fibroblast STO protein extracts and Miss Jessica Liu for helpful discussions and suggestions.

References

 

DC FieldValueLanguage
dc.contributor.authorKong, RPWen_HK
dc.contributor.authorSiu, SOen_HK
dc.contributor.authorLee, SSMen_HK
dc.contributor.authorLo, Cen_HK
dc.contributor.authorChu, IKen_HK
dc.date.accessioned2011-07-27T01:27:02Z-
dc.date.available2011-07-27T01:27:02Z-
dc.date.issued2011en_HK
dc.identifier.citationJournal Of Chromatography A, 2011, v. 1218 n. 23, p. 3681-3688en_HK
dc.identifier.issn0021-9673en_HK
dc.identifier.urihttp://hdl.handle.net/10722/135040-
dc.description.abstractPreviously, we described an online high-/low-pH RP-RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP-RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP-RP separation. Relative to the RP-RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample. © 2011 Elsevier B.V.en_HK
dc.languageengen_US
dc.publisherElsevier BV. The Journal's web site is located at http://www.elsevier.com/locate/chromaen_HK
dc.relation.ispartofJournal of Chromatography Aen_HK
dc.subjectHigh-/low-pH RP-RPen_HK
dc.subjectLiquid chromatographyen_HK
dc.subjectMDLCen_HK
dc.subjectProteomicsen_HK
dc.subjectRP-SCX-RPen_HK
dc.subjectTwo-dimensionalen_HK
dc.subject.meshArabidopsis Proteins - chemistry-
dc.subject.meshChromatography, Ion Exchange - methods-
dc.subject.meshChromatography, Reverse-Phase - methods-
dc.subject.meshPeptide Mapping - methods-
dc.subject.meshProteomics - methods-
dc.titleDevelopment of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approachen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0021-9673&volume=1218&issue=23&spage=3681&epage=3688&date=2011&atitle=Development+of+online+high-/low-pH+reversed-phase–reversed-phase+two-dimensional+liquid+chromatography+for+shotgun+proteomics:+a+reversed-phase-strong+cation+exchange-reversed-phase+approach-
dc.identifier.emailLo, C: clivelo@hkucc.hku.hken_HK
dc.identifier.emailChu, IK: ivankchu@hku.hken_HK
dc.identifier.authorityLo, C=rp00751en_HK
dc.identifier.authorityChu, IK=rp00683en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1016/j.chroma.2011.04.022en_HK
dc.identifier.pmid21531424-
dc.identifier.scopuseid_2-s2.0-79955951005en_HK
dc.identifier.hkuros186823en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-79955951005&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume1218en_HK
dc.identifier.issue23en_HK
dc.identifier.spage3681en_HK
dc.identifier.epage3688en_HK
dc.identifier.isiWOS:000291521200015-
dc.publisher.placeNetherlandsen_HK
dc.identifier.scopusauthoridKong, RPW=35217869000en_HK
dc.identifier.scopusauthoridSiu, SO=8603087200en_HK
dc.identifier.scopusauthoridLee, SSM=49963897600en_HK
dc.identifier.scopusauthoridLo, C=15737175700en_HK
dc.identifier.scopusauthoridChu, IK=7103327484en_HK
dc.identifier.citeulike9189359-

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