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Miniaturized on-line proteolysis-packed capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase
Mid Sweden University, Faculty of Science, Technology and Media, Department of Natural Sciences.ORCID iD: 0000-0002-3646-294X
2003 (English)In: Journal of Chromatography A, ISSN 0021-9673, Vol. 998, no 1/2, 83-91 p.Article in journal (Refereed) Published
Abstract [en]

In this study, methodology was developed for on-line and miniaturized enzymatic digestion with liquid chromatographic (LC) separation and mass spectrometric (MS) detection. A packed capillary LC–MS system was combined with on-line trypsin cleavage of a model protein, lactate dehydrogenase, to provide an efficient system for peptide mapping. The protein was injected onto an enzymatic capillary reactor and the resulting peptides were efficiently trapped on a capillary trapping column. Different trapping columns were evaluated to achieve a high binding capacity for the peptides generated in the enzyme reactor. The peptides were further eluted from the pre-column and separated on an analytical capillary column by a buffer more suitable for the following an electrospray ionisation (ESI) MS process. An important aspect of the on-line approach was the desalting of peptides performed in the trapping column to avoid detrimental signal suppression in the ESI process. The developed on-line system was finally compared to a classical digestion in solution, with reference to peptide sequence coverage and sensitivity. It was shown that the on-line system gave more than 100% higher peptide sequence coverage than traditional digestion methods.

Place, publisher, year, edition, pages
2003. Vol. 998, no 1/2, 83-91 p.
Keyword [en]
peptide mapping, proteolysis, lactate dehydrogenase, enzymes
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:miun:diva-2507DOI: 10.1016/S0021-9673(03)00647-2Local ID: 1761OAI: oai:DiVA.org:miun-2507DiVA: diva2:27539
Available from: 2008-09-30 Created: 2008-09-30 Last updated: 2011-01-10Bibliographically approved

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CiteExportLink to record
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Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
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Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
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Output format
  • html
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  • asciidoc
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