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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
Kong R.P.W.1; Siu S.O.1; Lee S.S.M.3; Lo C.2; Chu I.K.1
2011
Source PublicationJournal of Chromatography A
ISSN219673
Volume1218Issue:23Pages:3681
Abstract

Previously, 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.

KeywordHigh-/low-ph Rp-rp Liquid Chromatography Mdlc Proteomics Rp-scx-rp Two-dimensional
DOI10.1016/j.chroma.2011.04.022
URLView the original
Indexed BySCI
Language英语
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS SubjectBiochemical Research Methods ; Chemistry, Analytical
WOS IDWOS:000291521200015
The Source to ArticleScopus
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Cited Times [WOS]:40   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionInstitute of Chinese Medical Sciences
Corresponding AuthorLo C.; Chu I.K.
Affiliation1.Department of Chemistry, The University of Hong Kong, Hong Kong, China
2.School of Biological Sciences, The University of Hong Kong, Hong Kong, China
3.State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China
Recommended Citation
GB/T 7714
Kong R.P.W.,Siu S.O.,Lee S.S.M.,et al. 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[J]. Journal of Chromatography A,2011,1218(23):3681.
APA Kong R.P.W.,Siu S.O.,Lee S.S.M.,Lo C.,&Chu I.K..(2011).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.Journal of Chromatography A,1218(23),3681.
MLA Kong R.P.W.,et al."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".Journal of Chromatography A 1218.23(2011):3681.
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