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Article: Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications

TitleMicrowave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications
Authors
Issue Date2008
PublisherPergamon. The Journal's web site is located at http://www.elsevier.com/locate/carres
Citation
Carbohydrate Research, 2008, v. 343 n. 17, p. 2870-2877 How to Cite?
AbstractNonspecific proteolytic digestion of glycoproteins is an established technique in glycomics and glycoproteomics. In the presence of pronase E, for example, glycoproteins are digested to small glycopeptides having one to six amino acids residues, which can be analyzed with excellent sensitivity using mass spectrometry. Unfortunately, the long digestion times (1-3 days) limit the analytical throughput. In this study, we used controlled microwave irradiation to accelerate the proteolytic cleavage of glycoproteins mediated by pronase E. We used ESI-MS and MALDI-MS analyses to evaluate the microwave-assisted enzymatic digestions at various digestion durations, temperatures, and enzyme-to-protein ratios. When digesting glycoproteins, pronase E produced glycopeptides within 5 min under microwave irradiation; glycopeptides having one or two amino acids were the major products. Although analysis of peptides containing multiple amino acid residues offers the opportunity for peptide sequencing and provides information regarding the sites of glycosylation, the signals of Asn-linked glycans were often suppressed by the glycopeptides containing basic amino acids (Lys or Arg) in MALDI-MS experiments. To minimize this signal-to-content dependence, we converted the glycopeptides into their sodiated forms and then methylated them using methyl iodide. This controlled methylation procedure resulted in quaternization of the amino group of the N-terminal amino acid residue. Using this approach, the mass spectrometric response of glyco-Asn was enhanced, compensating for the poorer ionization efficiency associated with the basic amino acids residues. The methylated products of glycopeptides containing two or more amino acid residues were more stable than those containing only a single Asn residue. This feature can be used to elucidate glycan structures and glycosylation sites without the need for MS/MS analysis. Crown Copyright © 2008.
Persistent Identifierhttp://hdl.handle.net/10722/168333
ISSN
2015 Impact Factor: 1.817
2015 SCImago Journal Rankings: 0.612
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLiu, Xen_US
dc.contributor.authorChan, Ken_US
dc.contributor.authorChu, IKen_US
dc.contributor.authorLi, Jen_US
dc.date.accessioned2012-10-08T03:17:41Z-
dc.date.available2012-10-08T03:17:41Z-
dc.date.issued2008en_US
dc.identifier.citationCarbohydrate Research, 2008, v. 343 n. 17, p. 2870-2877en_US
dc.identifier.issn0008-6215en_US
dc.identifier.urihttp://hdl.handle.net/10722/168333-
dc.description.abstractNonspecific proteolytic digestion of glycoproteins is an established technique in glycomics and glycoproteomics. In the presence of pronase E, for example, glycoproteins are digested to small glycopeptides having one to six amino acids residues, which can be analyzed with excellent sensitivity using mass spectrometry. Unfortunately, the long digestion times (1-3 days) limit the analytical throughput. In this study, we used controlled microwave irradiation to accelerate the proteolytic cleavage of glycoproteins mediated by pronase E. We used ESI-MS and MALDI-MS analyses to evaluate the microwave-assisted enzymatic digestions at various digestion durations, temperatures, and enzyme-to-protein ratios. When digesting glycoproteins, pronase E produced glycopeptides within 5 min under microwave irradiation; glycopeptides having one or two amino acids were the major products. Although analysis of peptides containing multiple amino acid residues offers the opportunity for peptide sequencing and provides information regarding the sites of glycosylation, the signals of Asn-linked glycans were often suppressed by the glycopeptides containing basic amino acids (Lys or Arg) in MALDI-MS experiments. To minimize this signal-to-content dependence, we converted the glycopeptides into their sodiated forms and then methylated them using methyl iodide. This controlled methylation procedure resulted in quaternization of the amino group of the N-terminal amino acid residue. Using this approach, the mass spectrometric response of glyco-Asn was enhanced, compensating for the poorer ionization efficiency associated with the basic amino acids residues. The methylated products of glycopeptides containing two or more amino acid residues were more stable than those containing only a single Asn residue. This feature can be used to elucidate glycan structures and glycosylation sites without the need for MS/MS analysis. Crown Copyright © 2008.en_US
dc.languageengen_US
dc.publisherPergamon. The Journal's web site is located at http://www.elsevier.com/locate/carresen_US
dc.relation.ispartofCarbohydrate Researchen_US
dc.subject.meshAmino Acids - Analysisen_US
dc.subject.meshGlycomics - Methodsen_US
dc.subject.meshGlycopeptides - Chemistry - Radiation Effectsen_US
dc.subject.meshGlycoproteins - Chemistry - Radiation Effectsen_US
dc.subject.meshHydrolysisen_US
dc.subject.meshKineticsen_US
dc.subject.meshMethylationen_US
dc.subject.meshMicrowavesen_US
dc.subject.meshOligosaccharides - Chemistry - Radiation Effectsen_US
dc.subject.meshPeptides - Chemistry - Radiation Effectsen_US
dc.subject.meshPronaseen_US
dc.subject.meshProteomics - Methodsen_US
dc.subject.meshSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - Methodsen_US
dc.titleMicrowave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applicationsen_US
dc.typeArticleen_US
dc.identifier.emailChu, IK:ivankchu@hku.hken_US
dc.identifier.authorityChu, IK=rp00683en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1016/j.carres.2008.07.010en_US
dc.identifier.pmid18768173-
dc.identifier.scopuseid_2-s2.0-53849116073en_US
dc.identifier.hkuros155638-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-53849116073&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume343en_US
dc.identifier.issue17en_US
dc.identifier.spage2870en_US
dc.identifier.epage2877en_US
dc.identifier.isiWOS:000261134300003-
dc.publisher.placeUnited Kingdomen_US
dc.identifier.scopusauthoridLiu, X=36064967500en_US
dc.identifier.scopusauthoridChan, K=7406031631en_US
dc.identifier.scopusauthoridChu, IK=7103327484en_US
dc.identifier.scopusauthoridLi, J=25931590500en_US

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