title: Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts.
abstract: A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Hanne Kolsrud
authors.initials: HK
authors.lastname: Hustoft
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Tore
authors.initials: T
authors.lastname: Vehus
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Ole Kristian
authors.initials: OK
authors.lastname: Brandtzaeg
authors.affiliation: Unit for Cell Signaling, Cancer Stem Cell Innovation Center, Oslo University Hospital, Oslo, Norway.
authors.forename: Stefan
authors.initials: S
authors.lastname: Krauss
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Tyge
authors.initials: T
authors.lastname: Greibrokk
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Steven Ray
authors.initials: SR
authors.lastname: Wilson
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
mesh_terms: D015415:Biomarkers / Q000737:chemistry; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D051153:Wnt Proteins / Q000737:chemistry*
chemical_list: D015415:Biomarkers; D051153:Wnt Proteins