Integrated enzyme reactor and high resolving chromatography in “sub-chip” dimensions for sensitive protein mass spectrometry

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-...

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Veröffentlicht in:Scientific reports 2013-12, Vol.3 (1), p.3511-3511, Article 3511
Hauptverfasser: Hustoft, Hanne Kolsrud, Brandtzaeg, Ole Kristian, Rogeberg, Magnus, Misaghian, Dorna, Torsetnes, Silje Bøen, Greibrokk, Tyge, Reubsaet, Léon, Wilson, Steven Ray, Lundanes, Elsa
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Sprache:eng
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Zusammenfassung: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep03511