title: Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols.
abstract: Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000-80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1-21% and 2-20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Hanne
authors.initials: H
authors.lastname: Roberg-Larsen
authors.affiliation: Unit for Cell Signaling, Cancer Stem Cell Innovation Centre (SFI-CAST), Oslo University Hospital, Rikshospitalet, Oslo, Norway Affitech Research AS, Oslo, Norway.
authors.forename: Kaja
authors.initials: K
authors.lastname: Lund
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Tore
authors.initials: T
authors.lastname: Vehus
authors.affiliation: Unit for Cell Signaling, Cancer Stem Cell Innovation Centre (SFI-CAST), Oslo University Hospital, Rikshospitalet, Oslo, Norway.
authors.forename: Nina
authors.initials: N
authors.lastname: Solberg
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Caroline
authors.initials: C
authors.lastname: Vesterdal
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Dorna
authors.initials: D
authors.lastname: Misaghian
authors.affiliation: Unit for Cell Signaling, Cancer Stem Cell Innovation Centre (SFI-CAST), Oslo University Hospital, Rikshospitalet, Oslo, Norway.
authors.forename: Petter Angell
authors.initials: PA
authors.lastname: Olsen
authors.affiliation: Unit for Cell Signaling, Cancer Stem Cell Innovation Centre (SFI-CAST), Oslo University Hospital, Rikshospitalet, Oslo, Norway.
authors.forename: Stefan
authors.initials: S
authors.lastname: Krauss
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: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D000072376:Oxysterols / Q000032:analysis*
chemical_list: D000072376:Oxysterols
keywords: 24S hydroxycholesterol; 25 hydroxycholesterol; 27 hydroxycholesterol; cholesterol 24-hydroxylase; cholesterol 25-hydroxylase; cholesterol 27-hydroxylase