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S. Brockbank, V.F. Miller, M. Oliver, W.J. Curry; Selective Characterisation of the Porcine Neuroretinal Proteome . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4563.
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Purpose:One of the primary aims of biological research in the next decade will be to ascribe function to the thousands of proteins thus far revealed through the human genome-sequencing project. An estimated 500,000 proteins and peptides are resident in human tissues, however only a fraction of these are expressed in a cell and tissue type or bodily fluid at any given time. To understand the relevance of a protein to disease-related processes, it is important to register where, when, and to what extent a protein is expressed and the degree, if any, of post-translational processing. This area of research is called proteomics. This study employed differential protein solvation, allied with reverse phase HPLC (rpHPLC) and latterly electrospray ionisation quadrupole mass spectrometry (ESI-MS) to characterise and localise a defined quota of retinal proteins. Methods:Porcine retinae (n=20) were dissected and homogenised under liquid nitrogen prior to extraction. In brief, crushed retinae (5 g) were shaken overnight at 4 °C in 8 ml of acidified ethanol (pH 1.4). The sample was centrifuged, supernatant decanted, lyophilised and reconstituted in 0.1% (v/v) TFA/H20. Protein extracts were resolved to homogeneity using rpHPLC (absorbencies were measured at 214 nm (peptide bond) and 280 nm (aromatic residues)) employing a range of column chemistries (C4, C5, C8, C18). Homogeneous proteins were identified on a Micromass Q-TOF Ultima API tandem mass spectrometer. The distribution of a range of identified proteins within the neural retina were analysed employing immunohistochemistry and in situ hybridisation. Results:The extracted protein concentration was assessed using a commercially available protein estimation kit (PlusOne 2-D Quant Kit, Amersham). The extracted proteome was initially resolved using semi-preparative rpHPLC. The resultant prominent proteins profiles were successfully resolved to homogeneity employing a range of column chemistries allied with modulation of the hydrophobic mobile phase and with the application of distinct counterions. Homogeneous proteins were identified by mass spectrometry allied with genome data base analysis. Immunohistochemistry and in situ hybridisation localised protein products to a range of neural retinal cell types and plexiform layers. Conclusions:This study has revealed that the application of distinct protein solvation protocols allied with rpHPLC offers a mechanism to identify a range of protein products. rpHPLC offers an alternative proteome research platform to that of 2D electrophoresis. However, selective integrations of these two techniques increases proteome resolution.
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