Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

Lam, Yun W.; Evans, Vanessa C.; Heesom, Kate J.; Lamond, Angus I.; Matthews, David A.

LC-MSMS Analysis-Peptides were isolated from each gel slice after in-gel digestion, desalted, and concentrated as described previously (32); separated by HPLC (Agilent) on a C 18 reverse phase column; and analyzed by a QSTAR XL hybrid quadrupole TOF mass spectrometer (Applied Biosystems). The peak lists were generated by the Analyst QS software, version 1.1 (Applied Biosystems). The MS data were searched against the NCBInr database (February 19, 2006 release, 3,230,559 sequences searched) for Homo sapiens using the MASCOT search engine, version 1.9 (Matrix Science). Variable modifications used were carboxymethyl (Cys), oxidation (Met) and phospho (Ser, Thr, and Tyr) as well as the appropriate SILAC modifica-tions. Trypsin specificity was used, two missed cleavages were allowed, and a mass tolerance of 0.5 Da was used for both precursor and fragment ions. Peptide charges of Ï©1, Ï©2, and Ï©3 were selected. Individual ions with MASCOT scores higher than 20 were used, making sure the "average peptide scores" of all identified proteins exceeded 20, a threshold commonly used for confident protein identification from tandem MS data (36) . Only bold red peptides were also considered, effectively removed duplicate homologous proteins from the results. Under these conditions, the estimated false positive rate is less than 5%, according to a previous analysis (37) . SILAC quantitation was done by the MSQuant software, which measures the averaged MS peak areas of the isotopic pairs. Only proteins with bold red peptides and combined scores higher than 50 were quantitated. Proteins with heavy/light isotopic ratios lower than 0.01 were discarded; they mostly represented environmental contaminants. The S.D. of a protein ratio represented the variations among the measured peptide ratios for the same protein. Functional classification of the identified proteins was performed using Proteincenter (Proxeon). The biological reproducibility was addressed by a parallel 2D gel-based approach and by microscopy analysis (see below).