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- Publisher Website: 10.1016/j.carres.2008.07.010
- Scopus: eid_2-s2.0-53849116073
- PMID: 18768173
- WOS: WOS:000261134300003
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Article: Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications
Title | Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications |
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Authors | |
Keywords | Glycopeptides Glycosylation MALDI-TOF/TOF Mass spectrometry Microwave activation |
Issue Date | 2008 |
Publisher | Pergamon. 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? |
Abstract | Nonspecific 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 Identifier | http://hdl.handle.net/10722/168333 |
ISSN | 2023 Impact Factor: 2.4 2023 SCImago Journal Rankings: 0.509 |
ISI Accession Number ID | |
References |
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Liu, X | en_US |
dc.contributor.author | Chan, K | en_US |
dc.contributor.author | Chu, IK | en_US |
dc.contributor.author | Li, J | en_US |
dc.date.accessioned | 2012-10-08T03:17:41Z | - |
dc.date.available | 2012-10-08T03:17:41Z | - |
dc.date.issued | 2008 | en_US |
dc.identifier.citation | Carbohydrate Research, 2008, v. 343 n. 17, p. 2870-2877 | en_US |
dc.identifier.issn | 0008-6215 | en_US |
dc.identifier.uri | http://hdl.handle.net/10722/168333 | - |
dc.description.abstract | Nonspecific 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.language | eng | en_US |
dc.publisher | Pergamon. The Journal's web site is located at http://www.elsevier.com/locate/carres | en_US |
dc.relation.ispartof | Carbohydrate Research | en_US |
dc.subject | Glycopeptides | - |
dc.subject | Glycosylation | - |
dc.subject | MALDI-TOF/TOF | - |
dc.subject | Mass spectrometry | - |
dc.subject | Microwave activation | - |
dc.subject.mesh | Amino Acids - Analysis | en_US |
dc.subject.mesh | Glycomics - Methods | en_US |
dc.subject.mesh | Glycopeptides - Chemistry - Radiation Effects | en_US |
dc.subject.mesh | Glycoproteins - Chemistry - Radiation Effects | en_US |
dc.subject.mesh | Hydrolysis | en_US |
dc.subject.mesh | Kinetics | en_US |
dc.subject.mesh | Methylation | en_US |
dc.subject.mesh | Microwaves | en_US |
dc.subject.mesh | Oligosaccharides - Chemistry - Radiation Effects | en_US |
dc.subject.mesh | Peptides - Chemistry - Radiation Effects | en_US |
dc.subject.mesh | Pronase | en_US |
dc.subject.mesh | Proteomics - Methods | en_US |
dc.subject.mesh | Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - Methods | en_US |
dc.title | Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications | en_US |
dc.type | Article | en_US |
dc.identifier.email | Chu, IK:ivankchu@hku.hk | en_US |
dc.identifier.authority | Chu, IK=rp00683 | en_US |
dc.description.nature | link_to_subscribed_fulltext | en_US |
dc.identifier.doi | 10.1016/j.carres.2008.07.010 | en_US |
dc.identifier.pmid | 18768173 | - |
dc.identifier.scopus | eid_2-s2.0-53849116073 | en_US |
dc.identifier.hkuros | 155638 | - |
dc.relation.references | http://www.scopus.com/mlt/select.url?eid=2-s2.0-53849116073&selection=ref&src=s&origin=recordpage | en_US |
dc.identifier.volume | 343 | en_US |
dc.identifier.issue | 17 | en_US |
dc.identifier.spage | 2870 | en_US |
dc.identifier.epage | 2877 | en_US |
dc.identifier.isi | WOS:000261134300003 | - |
dc.publisher.place | United Kingdom | en_US |
dc.identifier.scopusauthorid | Liu, X=36064967500 | en_US |
dc.identifier.scopusauthorid | Chan, K=7406031631 | en_US |
dc.identifier.scopusauthorid | Chu, IK=7103327484 | en_US |
dc.identifier.scopusauthorid | Li, J=25931590500 | en_US |
dc.identifier.issnl | 0008-6215 | - |