NDLI: 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

Content Provider	Royal Society of Chemistry (RSC)
Author	Lothian, Amber Hare, Dominic J. Cherny, Robert A. Liddell, Jeffrey R. Masters, Colin L. Grimm, Rudolf Bush, Ashley I. Roberts, Blaine R. Doble, Philip A. White, Anthony R. Grubman, Alexandra Matsuda, Toshiaki Ryan, Timothy M.
Copyright Year	2013
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.
Starting Page	1656
Ending Page	1662
Page Count	7
File Format	HTM / HTML PDF
ISSN	17565901
Volume Number	5
Issue Number	12
Journal	Metallomics
DOI	10.1039/c3mt00227f
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Western blot Cu Neuron Metalloprotein Ferritin Superoxide dismutase Astrocyte Protein
Content Type	Text
Resource Type	Article
Subject	Chemistry Medicine Metals and Alloys Biochemistry Biomaterials Biophysics

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