- Title
- Mass spectrometry measurement of metals and metalloproteins in brain tissue
- Creator
- Paul, Mitchell
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2012
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Metal ions carry out a number of essential processes in physiological functions including neuronal signalling, gene expression and catalysis. Increasing attention towards the roles that metals play has been largely driven by their implication in neurological disorders such as ALS, AD and PD. While systematic studies have been carried out on the CNS in the diseased state few have been performed on the same healthy tissues in a standardized fashion. Furthermore, there is a paucity of quantitative data concerning metal distribution during neural re-organization and plasticity. The normal distribution of a range of elements in the brain tissue of healthy Wistar rats was established using Inductively Coupled Plasmas Sector Field Mass Spectrometry (ICP-SF-MS). A method was developed to determine concentrations of Ag, Cd, Hg, Pb, Bi, U, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As and Se in specific brain regions. A principal component analysis was then used to reveal elemental patterns of the brain regions. This standardized method was used in subsequent investigations of the barrel cortex thereby allowing direct comparisons to be made given that the sampling, sample preparation and instrument operation were performed under the same analytical conditions. In contrast, most “reference values” for trace element content in the CNS of “normal” populations gather results from disparate studies performed by researchers using distinct techniques and a wide variety of instruments. The fluctuation of Zn was investigated in the barrel cortex of rats contralateral and ipsilateral to partial vibrissae removal (checkerboard pattern plucking). Quantitative analysis was carried out using ICP-SF-MS. This allowed measurement of total Zn content and separated low molecular weight (free) and protein bound components. A significant decrease of Zn at 8 h after whisker plucking was revealed in both deprived and non-deprived barrel cortices. Thereafter, zinc content increased above control levels for the length of the study (up to 96 h), confirming previous studies using histochemical staining methods for free zinc. All changes are attributable to free Zn as no change was observed in the protein bound form as sampled at 8 and 24 hr post manipulation. From the same study additional data is presented on the fluctuation of metalloproteins and unbound metals in the barrel cortex after vibrissae removal. Copper, Mn, Fe and Mg fluctuation were investigated in the barrel cortex of rats with subsequent analysis using ICP-SF-MS and Electrospray Ionization Time-of-Flight Mass Spectrometry (ESI-TOF-MS) for metal quantification and protein identification, respectively. A significant decrease of Mn and Cu at 8 h after whisker plucking was revealed in both deprived and non-deprived barrel cortices. Changes to Cu were attributable to protein-bound species and changes to Mn were attributed to both protein-bound and non-protein bound species. The significant decrease of the metals Cu and Mn (along with Zn) at 8 h post-vibrissectomy indicated a synchronised compensatory mechanism of the CNS for reduced sensory input given their role in modulation of neural activity. In addition, several proteins of interest were identified for further investigation demonstrating the utility of ICP-SF-MS as a screening technique for metalloproteins. Coupled with high resolution separation of the protein, ICP-SF-MS offers itself as an innovative analytical tool. Given that approximately a third of proteins require a metal cofactor, a systematic approach to the study of metal content, speciation, localization and use within the CNS is supported.
- Subject
- ICP-MS; trace metals; metalloproteins; brain tissue
- Identifier
- http://hdl.handle.net/1959.13/936121
- Identifier
- uon:12219
- Rights
- Copyright 2012 Mitchell Paul
- Language
- eng
- Full Text
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| View Details Download | ATTACHMENT01 | Abstract | 29 KB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Thesis | 2 MB | Adobe Acrobat PDF | View Details Download |