Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins - Nature Biotechnology

]-arginine . Cells were maintained in

medium for five passages to ensure complete isotopic labeling. Cells were then treated with either PBS control or 100 nM bispecific for 48 h before cells were collected, and heavy/light-labeled cells were mixed at a 1:1 ratio in both forward and reverse mode. A small portion of these cells was set aside for whole-cell proteomic analysis, and the remainder was used to prepare surface-proteome enrichment.but using a modified protocol to facilitate small sample input.

proteomics data were analyzed using PEAKSOnline . For all samples, searches were performed with a precursor mass error tolerance of 20 ppm and a fragment mass error tolerance of 0.03 Da. The digest was considered semispecific, and up to three missed cleavages were allowed. For whole-cell proteome data, the reviewed SwissProt database for the human proteome was used.

analysis was performed using the forward and reverse samples, and at least two labels for the ID and features were required. Proteins showing a greater than twofold change from PBS control with a significance ofCell viability assays were performed using an MTT modified assay. Briefly, on day 0, 15,000 MDA-MB-175VII or 7,000 NCI-H358 cells were plated in each well of a 96-well plate. On day 1, bispecifics or control antibodies were added in a dilution series.

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