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Article: Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization

TitleOnline coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization
Authors
KeywordsFull-component analysis
Glycan separation
Hydrophilic analytes
RP-HILIC
UPLC
Issue Date2010
PublisherSpringer Verlag. The Journal's web site is located at http://link.springer.de/link/service/journals/00216/index.htm
Citation
Analytical And Bioanalytical Chemistry, 2010, v. 398 n. 2, p. 791-804 How to Cite?
AbstractWe have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC-with only a single sample injection event-as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans. © 2010 The Author(s).
Persistent Identifierhttp://hdl.handle.net/10722/124006
ISSN
2015 Impact Factor: 3.125
2015 SCImago Journal Rankings: 1.096
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
Hong Kong Research Grant CouncilHKU7640/07M
HKU7650/09P
HKU1/07C
Hong Kong Special Administrative Region, China
HKU200811159027
Funding Information:

This study was supported by the Hong Kong Research Grant Council (Project Nos. HKU7640/07M, HKU7650/09P, and HKU1/07C), Hong Kong Special Administrative Region, China, and the HKU seed funding program (200811159027). We thank Dr. W. L. Chan for providing the mouse chondrogenic cell protein extracts.

References

 

DC FieldValueLanguage
dc.contributor.authorLam, MPYen_HK
dc.contributor.authorSiu, SOen_HK
dc.contributor.authorLau, Een_HK
dc.contributor.authorMao, Xen_HK
dc.contributor.authorSun, HZen_HK
dc.contributor.authorChiu, PCNen_HK
dc.contributor.authorYeung, WSBen_HK
dc.contributor.authorCox, DMen_HK
dc.contributor.authorChu, IKen_HK
dc.date.accessioned2010-10-19T04:32:34Z-
dc.date.available2010-10-19T04:32:34Z-
dc.date.issued2010en_HK
dc.identifier.citationAnalytical And Bioanalytical Chemistry, 2010, v. 398 n. 2, p. 791-804en_HK
dc.identifier.issn1618-2642en_HK
dc.identifier.urihttp://hdl.handle.net/10722/124006-
dc.description.abstractWe have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC-with only a single sample injection event-as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans. © 2010 The Author(s).en_HK
dc.languageengen_HK
dc.publisherSpringer Verlag. The Journal's web site is located at http://link.springer.de/link/service/journals/00216/index.htmen_HK
dc.relation.ispartofAnalytical and Bioanalytical Chemistryen_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong Licenseen_HK
dc.rightsThe original publication is available at www.springerlink.com-
dc.subjectFull-component analysisen_HK
dc.subjectGlycan separationen_HK
dc.subjectHydrophilic analytesen_HK
dc.subjectRP-HILICen_HK
dc.subjectUPLCen_HK
dc.subject.meshAmino Acid Sequence-
dc.subject.meshChromatography, Reverse-Phase - instrumentation - methods-
dc.subject.meshGlycoproteins - analysis - isolation and purification-
dc.subject.meshProteins - analysis - isolation and purification-
dc.subject.meshProteomics - instrumentation - methods-
dc.titleOnline coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterizationen_HK
dc.typeArticleen_HK
dc.identifier.emailSun, HZ:hsun@hkucc.hku.hken_HK
dc.identifier.emailChiu, PCN:pchiucn@hku.hken_HK
dc.identifier.emailYeung, WSB:wsbyeung@hkucc.hku.hken_HK
dc.identifier.emailChu, IK:ivankchu@hku.hken_HK
dc.identifier.authoritySun, HZ=rp00777en_HK
dc.identifier.authorityChiu, PCN=rp00424en_HK
dc.identifier.authorityYeung, WSB=rp00331en_HK
dc.identifier.authorityChu, IK=rp00683en_HK
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.1007/s00216-010-3991-2en_HK
dc.identifier.pmid20632160-
dc.identifier.pmcidPMC2939347-
dc.identifier.scopuseid_2-s2.0-77957883394en_HK
dc.identifier.hkuros186817en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-77957883394&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume398en_HK
dc.identifier.issue2en_HK
dc.identifier.spage791en_HK
dc.identifier.epage804en_HK
dc.identifier.eissn1618-2650en_HK
dc.identifier.isiWOS:000281842400017-
dc.publisher.placeGermanyen_HK
dc.description.otherSpringer Open Choice, 01 Dec 2010-
dc.identifier.scopusauthoridLam, MPY=35302594800en_HK
dc.identifier.scopusauthoridSiu, SO=8603087200en_HK
dc.identifier.scopusauthoridLau, E=35302963200en_HK
dc.identifier.scopusauthoridMao, X=7402841292en_HK
dc.identifier.scopusauthoridSun, HZ=7404827446en_HK
dc.identifier.scopusauthoridChiu, PCN=25959969200en_HK
dc.identifier.scopusauthoridYeung, WSB=7102370745en_HK
dc.identifier.scopusauthoridCox, DM=35434137900en_HK
dc.identifier.scopusauthoridChu, IK=7103327484en_HK

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