Extended Data Fig. 3: Coverage Improvement of SEE-CITE Workflow and Target Identification.

A, SEE-CITE AfBPP workflow for comparing C3 and C4 biotin coverage. B, Assessing the impact of scaling protein input, sample cleanup by SP3 versus chloroform/methanol and extended gradient on coverage of SEE-CITE-modified peptides identified for 2b (20 μM, 1 h) in K562 cells. Data are mean ± SD (n = 3 biological replicates for entry 1, 3, and ‘optimized,’ n = 2 biological replicates for entry 2, 4-6). Best ‘optimized’ conditions reflect samples prepared using conditions from entry 6 with acquisition using a 140 min gradient. C, Optimization of MS2 resolving power (n = 1). The sample in entry 4, Fig. 2c, was used for this analysis. D, Comparison of different low-retention plastic containers, Vendor A (Fisher #02-681-320) and Vendor B (Sarstedt #72.706.600) (n = 1). E, Coverage comparison of SEE-CITE-labeled peptides undergoing DTT reduction at 37 °C (30 mins) versus 65 °C (15 mins) (n = 1). F, Alternative SEE-CITE workflow combined with a clickable biotin capture reagent, sCIP, with a chemically cleavable dialkoxydiphenylsilane (DADPS) linker. G, Coverage comparison of peptide-labeled by SEE-CITE with biotin-azide and SEE-CITE paired with sCIP reagent. Data presented are mean ± SD n = 2 biological replicates. H, I, Mapping sites of labeling by 2a (20 μM, 1 h) in ‘D’ for ‘H’ human FKBP1A (PDB: 1J4H) and ‘I’ cathepsin D (PDB: 4OD9, (n = 2 biological replicates). All MS data can be found in Supplementary Table 2.

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