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Integrated enzyme reactor and high resolving chromatography in "sub-chip" dimensions for sensitive protein mass spectrometry.
Hustoft, Hanne Kolsrud; Brandtzaeg, Ole Kristian; Rogeberg, Magnus; Misaghian, Dorna; Torsetnes, Silje Bøen; Greibrokk, Tyge; Reubsaet, Léon; Wilson, Steven Ray; Lundanes, Elsa.
Afiliación
  • Hustoft HK; Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
  • Brandtzaeg OK; Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
  • Rogeberg M; 1] Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway [2] Department of Neurology, Akershus University Hospital, 1478 Lørenskog, Norway.
  • Misaghian D; Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
  • Torsetnes SB; School of Pharmacy, University of Oslo, Post Box 1068 Blindern, NO-0316 Oslo, Norway.
  • Greibrokk T; Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
  • Reubsaet L; School of Pharmacy, University of Oslo, Post Box 1068 Blindern, NO-0316 Oslo, Norway.
  • Wilson SR; Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
  • Lundanes E; Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
Sci Rep ; 3: 3511, 2013 Dec 16.
Article en En | MEDLINE | ID: mdl-24336509
ABSTRACT
Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using "sub-chip" columns (10-20 µm inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20 µm ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10 µm ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3-5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Espectrometría de Masas / Cromatografía Liquida / Proteómica / Enzimas Inmovilizadas Tipo de estudio: Diagnostic_studies Límite: Humans Idioma: En Revista: Sci Rep Año: 2013 Tipo del documento: Article País de afiliación: Noruega

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Espectrometría de Masas / Cromatografía Liquida / Proteómica / Enzimas Inmovilizadas Tipo de estudio: Diagnostic_studies Límite: Humans Idioma: En Revista: Sci Rep Año: 2013 Tipo del documento: Article País de afiliación: Noruega