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1、DATASETBRIEF Elucidation of the avian nucleolar proteome by quantitative proteomics using SILAC and changes in cells infected with the coronavirus infectious bronchitis virus Edward Emmott1,2, Catriona Smith1, Stevan R. Emmett3, Brian K. Dove4 and Julian A. Hiscox1,2 1 Institute of Molecular and Cel
2、lular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK 2 Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK 3 Clinical Sciences Building, CSRI, University Hospital Coventry and Warwickshire, Coventry, UK 4 Centre for Emergency Preparedness and Resp
3、onse, Health Protection Agency, Porton Down, Salisbury, UK Received: March 4, 2010 Revised: June 14, 2010 Accepted: July 25, 2010 The nucleolus is a dynamic subnuclear compartment involved in ribosome subunit biogen- esis, regulation of cell stress and modulation of cellular growth and the cell cycl
4、e, among other functions. The nucleolus is composed of complex protein/protein and protein/RNA interactions. It is a target of virus infection with many viral proteins being shown to localize to the nucleolus during infection. Perturbations to the structure of the nucleolus and its proteome have bee
5、n predicted to play a role in both cellular and infectious disease. Stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS with bioinformatic analysis using Ingenuity Pathway Analysis was used to investigate whether the nucleolar proteome altered in virus-infected cells. In thi
6、s study, the avian nucleolar proteome was defi ned in the absence and presence of virus, in this case the positive strand RNA virus, avian coronavirus infectious bronchitis virus. Data sets, potential protein changes and the func- tional consequences of virus infection were validated using independe
7、nt assays. These demonstrated that specifi c rather than generic changes occurred in the nucleolar proteome in infectious bronchitis virus-infected cells. Keywords: Avian coronavirus infectious bronchitis virus / Cell cycle / Microbiology / Protein complexes / Stable isotope labeling with amino acid
8、s The nucleolus is a subnuclear structure formed from complex proteinprotein and proteinnucleic acid interac- tions 1. The primary function of the nucleolus is in ribo- some biogenesis, regulation of the cell cycle and the response to cell stress 2. The nucleolus may be formed around hub proteins 1,
9、 depletion of which can result in alterations in nucleolar architecture 3 and possibly func- tion. The roles the nucleolus play outside its traditional function in ribosome biogenesis have only recently come to light, due in a large part to MS-based approaches to eluci- dating the nucleolar proteome
10、. The fi rst studies of the human nucleolar proteome revealed 271 and 213 proteins, respectively 4, 5. The most recent edition of the nucleolar proteomedatabase(NOPdb3.0) identifi edover4500 proteins as being resident within the nucleolus under certain conditions; it estimates at least 80% coverage
11、6. It is noteworthy that the majority of studies of the nucleolar proteome have been carried out on human HeLa cells, whereas the only other species investigated in depth is Arabidopsis thaliana where 217 proteins with a large degree Abbreviations: IBV, infectious bronchitis virus; N, nucleocapsid;
12、SILAC, stable isotope labeling with amino acids in cell culture Correspondence: Dr. Julian A. Hiscox, Institute of Molecular and Cellular Biology, Garstang Building, Room 8.58, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK E-mail: j.a.hiscoxleeds.ac.uk Fax: 144-1133433167 M
13、atrix Science, London, UK) against a concatenated database combining 80412 proteins from International Protein Index human protein database version 3.6 (forward database), and the reversed sequences of all proteins (reverse database). The complete methodology has been described previously 20, 22. Ra
14、w data sets used to generate Supporting Infor- mation Tables 1 and 2 (described below) can be found on the PRoteomics IDEntifi cations (PRIDE) database 23 and were uploaded using the PRIDE database convertor tool 24. Cellular proteins were initially assigned by comparing with the avian genome databa
15、se, and identifi ed 835 proteins (Supporting Information Table 1). However, this database had poor annotation compared with the human genome, and many of the proteins were unassigned or uncharacter- ized. Therefore, the human database was used for protein assignment from peptide identifi cation. In
16、addition, candi- date proteins were then examined to determine whether they were previously identifi ed in the human nucleolar proteome database, which contains some 4500 proteins with an estimated 80% coverage. Only if both of these criteria were met was the protein then assigned as being present in the avian nucleolar proteome (Supporting Information Table 2). These resulted in a fi nal list of 378 cellular proteins being identifi ed and of these 260 proteins being identifi ed and quantifi ed
