title: Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry.
abstract: Metals often determine the chemical reactivity of the proteins to which they are bound. Each cell in the body tightly maintains a unique metalloproteomic profile, mostly dependent on function. This paper describes an analytical online flow injection quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method, which was applied to profiling the metal-binding proteins found in primary cultures of neurons and astrocytes. This method can be conducted using similar amounts of sample to those used for Western blotting (20-150 μg protein), and has a turnaround time of <15 minutes. Metalloprotein standards for Fe (as ferritin), Cu and Zn (as superoxide dismutase-1) were used to construct multi-point calibration curves for online quantification of metalloproteins by SEC-ICP-MS. Homogenates of primary neuron and astrocyte cultures were analysed by SEC-ICP-MS. Online quantification by external calibration with metalloprotein standards determined the mass of metal eluting from the column relative to time (as pg s(-1)). Total on-column Fe, Cu and Zn detection limits ranged from 0.825 ± 0.005 ng to 13.6 ± 0.7 pg. Neurons and astrocytes exhibited distinct metalloprotein profiles, featuring both ubiquitous and unique metalloprotein species. Separation and detection by SEC-ICP-MS allows appraisal of these metalloproteins in their native state, and online quantification was achieved using this relatively simple external calibration process.
authors.affiliation: The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Kenneth Myer Building, 30 Royal Parade, Parkville, Victoria 3052, Australia. blaine.roberts@florey.edu.au.
authors.forename: Dominic J
authors.initials: DJ
authors.lastname: Hare
authors.forename: Alexandra
authors.initials: A
authors.lastname: Grubman
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Ryan
authors.forename: Amber
authors.initials: A
authors.lastname: Lothian
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Liddell
authors.forename: Rudolf
authors.initials: R
authors.lastname: Grimm
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Matsuda
authors.forename: Philip A
authors.initials: PA
authors.lastname: Doble
authors.forename: Robert A
authors.initials: RA
authors.lastname: Cherny
authors.forename: Ashley I
authors.initials: AI
authors.lastname: Bush
authors.forename: Anthony R
authors.initials: AR
authors.lastname: White
authors.forename: Colin L
authors.initials: CL
authors.lastname: Masters
authors.forename: Blaine R
authors.initials: BR
authors.lastname: Roberts
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000737:chemistry*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003300:Copper / Q000032:analysis*; D007501:Iron / Q000032:analysis*; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000737:chemistry*; D051379:Mice; D009474:Neurons / Q000737:chemistry*; D040901:Proteomics; D015032:Zinc / Q000032:analysis*
chemical_list: D008667:Metalloproteins; D003300:Copper; D007501:Iron; D015032:Zinc