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Article: Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples

TitleOnline combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples
Authors
KeywordsGlycoproteomics
LC
PGC
Proteomics
RP-RP
Issue Date2011
PublisherWiley - V C H Verlag GmbH & Co KGaA.
Citation
Electrophoresis, 2011, v. 32 n. 21, p. 2930-2940 How to Cite?
AbstractIn this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Persistent Identifierhttp://hdl.handle.net/10722/144518
ISSN
2015 Impact Factor: 2.482
2015 SCImago Journal Rankings: 0.896
ISI Accession Number ID
Funding AgencyGrant Number
Hong Kong Research Grants Council, Hong Kong Special Administrative Region, ChinaHKU7018/09P
HKU3/07C
Hong Kong RGC
Funding Information:

This study was supported by the Hong Kong Research Grants Council (project nos. HKU7018/09P and HKU3/07C), Hong Kong Special Administrative Region, China. E. L., M. P. Y. L., and D. C. M. N. thank the Hong Kong RGC for supporting their studentships. They thank Drs. Ronald Pang and Philip C. N. Chiu for providing the STO and BeWo Cell lysates.

References

 

DC FieldValueLanguage
dc.contributor.authorLam, MPYen_HK
dc.contributor.authorLau, Een_HK
dc.contributor.authorSiu, SOen_HK
dc.contributor.authorNg, DCMen_HK
dc.contributor.authorKong, RPWen_HK
dc.contributor.authorChiu, PCNen_HK
dc.contributor.authorYeung, WSBen_HK
dc.contributor.authorLo, Cen_HK
dc.contributor.authorChu, IKen_HK
dc.date.accessioned2012-02-03T06:12:02Z-
dc.date.available2012-02-03T06:12:02Z-
dc.date.issued2011en_HK
dc.identifier.citationElectrophoresis, 2011, v. 32 n. 21, p. 2930-2940en_HK
dc.identifier.issn0173-0835en_HK
dc.identifier.urihttp://hdl.handle.net/10722/144518-
dc.description.abstractIn this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.en_HK
dc.languageengen_US
dc.publisherWiley - V C H Verlag GmbH & Co KGaA.en_US
dc.relation.ispartofElectrophoresisen_HK
dc.subjectGlycoproteomicsen_HK
dc.subjectLCen_HK
dc.subjectPGCen_HK
dc.subjectProteomicsen_HK
dc.subjectRP-RPen_HK
dc.subject.meshChromatography, Reverse-Phase - instrumentation - methods-
dc.subject.meshGlycomics - methods-
dc.subject.meshGlycopeptides - analysis - chemistry - isolation and purification-
dc.subject.meshGraphite - chemistry-
dc.subject.meshProteomics - methods-
dc.titleOnline combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samplesen_HK
dc.typeArticleen_HK
dc.identifier.emailChiu, PCN: pchiucn@hku.hken_HK
dc.identifier.emailYeung, WSB: wsbyeung@hkucc.hku.hken_HK
dc.identifier.emailLo, C: clivelo@hkucc.hku.hken_HK
dc.identifier.emailChu, IK: ivankchu@hku.hken_HK
dc.identifier.authorityChiu, PCN=rp00424en_HK
dc.identifier.authorityYeung, WSB=rp00331en_HK
dc.identifier.authorityLo, C=rp00751en_HK
dc.identifier.authorityChu, IK=rp00683en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1002/elps.201100092en_HK
dc.identifier.pmid22009802-
dc.identifier.scopuseid_2-s2.0-80054984666en_HK
dc.identifier.hkuros198177en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-80054984666&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume32en_HK
dc.identifier.issue21en_HK
dc.identifier.spage2930en_HK
dc.identifier.epage2940en_HK
dc.identifier.isiWOS:000298101000002-
dc.publisher.placeGermanyen_HK
dc.identifier.scopusauthoridLam, MPY=35302594800en_HK
dc.identifier.scopusauthoridLau, E=35302963200en_HK
dc.identifier.scopusauthoridSiu, SO=8603087200en_HK
dc.identifier.scopusauthoridNg, DCM=36981534500en_HK
dc.identifier.scopusauthoridKong, RPW=35217869000en_HK
dc.identifier.scopusauthoridChiu, PCN=25959969200en_HK
dc.identifier.scopusauthoridYeung, WSB=7102370745en_HK
dc.identifier.scopusauthoridLo, C=15737175700en_HK
dc.identifier.scopusauthoridChu, IK=7103327484en_HK

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