title: Integrated enzyme reactor and high resolving chromatography in "sub-chip" dimensions for sensitive protein mass spectrometry.
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.
authors: Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
authors: Hanne Kolsrud
authors: HK
authors: Hustoft
authors: Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
authors: Ole Kristian
authors: OK
authors: Brandtzaeg
authors: 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.
authors: Magnus
authors: M
authors: Rogeberg
authors: Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
authors: Dorna
authors: D
authors: Misaghian
authors: School of Pharmacy, University of Oslo, Post Box 1068 Blindern, NO-0316 Oslo, Norway.
authors: Silje Bøen
authors: SB
authors: Torsetnes
authors: Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
authors: Tyge
authors: T
authors: Greibrokk
authors: School of Pharmacy, University of Oslo, Post Box 1068 Blindern, NO-0316 Oslo, Norway.
authors: Léon
authors: L
authors: Reubsaet
authors: Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
authors: Steven Ray
authors: SR
authors: Wilson
authors: Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
authors: Elsa
authors: E
authors: Lundanes
mesh_terms: D015415:Biomarkers; D002853:Chromatography, Liquid / Q000379:methods*; D004800:Enzymes, Immobilized; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D036103:Nanotechnology; D010446:Peptide Fragments / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry; D014357:Trypsin
chemical_list: D015415:Biomarkers; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011994:Recombinant Proteins; C087079:pro-gastrin-releasing peptide (31-98); D014357:Trypsin