Sub-proteome processing: isolation of neuromelanin granules from the human brain.
The sub-proteome analysis of organelles is a field of high relevance for molecular biology, because it provides detailed insights into the protein composition of cellular compartments. This approach not only results in a catalogue of organellar proteins, but in fact holds the potential to uncover the enzymatic armament engaged in biochemical reactions and to identify novel mechanisms of organelle biogenic pathways. Knowledge about protein localization may be a first step towards extensive functional analyses of specific target proteins engaged in development, aging, or disease. Moreover, several disorders of the human brain include aberrant protein function in specific compartments. Thus, a closer look at cellular organelles will allow for advancing our current perceptions of pathogenic processes. This chapter aims to provide a methodological workflow given by the isolation of neuromelanin granules from the human midbrain. This approach encompasses several modular steps that can easily be adjusted to any other organelle of interest and follows the sequence of (1) organelle isolation, (2) isolation quality controls by transmission electron microscopy and Western immuno blotting, and (3) gel-based protein separation towards protein identification by mass spectrometry.
Tribl
Florian
F
Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany. florian.tribl@gmx.de
D001921:Brain / Q000166:cytology; D001923:Brain Chemistry; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000648:ultrastructure; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008543:Melanins / Q000032:analysis* / Q000302:isolation & purification; D015388:Organelles / Q000737:chemistry / Q000648:ultrastructure; D020543:Proteome / Q000032:analysis*
D008543:Melanins; D020543:Proteome; C014121:neuromelanin