To more fully explore which proteins are modified with ISG15 in cell culture, we will undertake a more comprehensive screen using mass-spectroscopy – applying a strategy previously used to identify targets of the ubiquitin-like SUMO protein. TAP-tagging approaches coupled with MALDI-TOF mass spectrometry will be used. We will transfect cells with TAP-tagged or SPA-tagged ISG15 and purify the protein and protein complexes from cells treated or untreated with IFN. Using established procedures, we will then purify and identify the substrates using gel electrophoresis and mass spectrometry. Gel slices containing the proteins will be reductively alkylated and subjected to trypsinolysis. The peptides will be purified and analyzed by MALDI-TOF mass spectrometry using a cyano-4-hydroxycin-namic acid matrix on a Voyager DE-STR instrument (Applied Biosystems). Identification of the proteins using these mass fingerprinting data will be carried out using the ProFound software . Since MS cannot be used to quantify protein amounts, we will identify proteins of interest by comparing lists of proteins ISGylated under the various conditions and then perform Western blot studies to compare protein amounts. In doing these experiments we will use the MS equipment and expertise readily available at the Best Institute.