Fig. 7

LC-MS/MS-based identification of the ~ 25–30 kDa SH bands in rat glioma and bone marrow-derived mononuclear cells. Gel-ABPP was conducted using rat glioma homogenates (rGlioma) or lysates of rat bone marrow-derived mononuclear cells (rBM) as detailed in Materials and Methods. To facilitate SDS-PAGE separation of proteins with similar size, the proteomes (4 mg/ml) were deglycosylated (+) or underwent control treatment (−) prior to TAMRA-FP labeling. For deglycosylation, samples were treated with Protein Deglycosylation Mix II (New England Biolabs, Cat#P6044S) for 1 h at RT as per kit instructions, after which TAMRA-FP labeling was conducted using routine gel-ABPP protocol. Following in-gel fluorescence imaging, gel-pieces encompassing the SH bands of interest (numbered 1-15x) were cut and subjected to LC-MS/MS analysis, as described in Materials and Methods. Gel-pieces marked with x were cut as one sample containing also the numbered gel-piece and split thereafter, yielding two separate samples that were subjected to LC-MS/MS. The SHs identified from the glioma samples (1-7x) are listed in the middle (blue-white table) and those identified from rBM (8-15x) at right (yellow-white table). Note presence of the NSPs CTSG (Ctsg), NE (elastase 2, Elane) and PR3 (Prtn3) in the ~ 25 kDa gel-pieces of both proteomes. Note also presence of platelet-activating factor acetylhydrolases 1b2 and 1b3 (PAFAH1b3 and PAFAH1b2) in the 25–30 kDa gel-pieces of both proteomes. The complete LC-MS/MS data listing all identified proteins and additionally data on BT4C glioma cells is available as Supplementary File 1