Geneva Proteomics Centre: Research activities
Our goal is to develop, improve and automate protein identification and characterization methodologies using 2-DE, mass spectrometry and bioinformatics and to further apply them in the identification of specific disease associated markers and new therapeutic targets.
Nucleolar protein project
Nucleoli are dynamic, non-membrane separated nuclear domains, in which ribosomal genes are transcribed and ribosomal biosynthesis occurs. It has been proposed recently that nucleoli may play many other roles in several key events in life cycle by being a site of transient sequestration of cellular factors, such as cell cycle regulation, aging and telomerase maintenance, regulation of gene expression and possibly mRNA export. Indeed, the protein composition of nucleoli is yet not well characterised, though the first proteomic analysis were published on SWISS-2DPAGE and by Andersen et al (Current Biology 2002, vol. 12, 1-11). Furthermore, identification of proteins involved in newly proposed functions turns out to be difficult due to the presence of high amounts of ribosomal and structural proteins. Mass spectrometry has demonstrated its ability to rapidly identify a large number of proteins and became the heart of proteomic projects. In the present study, SDS-PAGE separation of nucleolar proteins purified from human HeLa cells were investigated by direct coupling of tandem mass spectrometry with nano-liquid-chromatography (nanoLC). After separation of 15 micrograms of nucleolar proteins by SDS-PAGE, the obtained gel was cut in 108 bands, proteins of each band were digested in gel and extracted peptides analysed by nanoLC-ESI-Q-TOF. Using this technique, 190 different proteins were identified, and up to 15 different proteins were found in one single band. Analysis of 45 spots from Coomassie blue stained 2-DE gel allowed to identify 23 additional proteins. Of all identified proteins, only about 50 % exhibit at least one known biological function. Sequence analysis using BLAST programs permitted to attribute hypothetical functions for half of the remaining 50 %. Finally, about 30 % of all identified proteins stand with an unpredictable function. As expected, a majority of known identified proteins are involved in ribosomal biogenesis. Numerous other identified proteins support the implication of nucleoli in other biological functions, such as translation regulation and export of mRNAs. Surprisingly, proteins with functions never related to the nucleolar structure were also found. Further studies on protein function are necessary to unravel these biological pathways.
Last modified 18/Sep/2002 by CHH
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