We performed initial gel-ABPP experiments to benchmark the proteome-wide reactivity of the alkynylated 3-amino stereoprobes 36A-D in comparison to the previously described 3-aryl stereoprobes MY-11A/B and MY-12A/B (Figure ). Exposure of the Ramos human B-cell cancer line to alkynylated stereoprobes (20 or 50 μM, 1 or 3 h), followed by lysis and copper-catalyzed azide-alkyne cyclo-addition (CuAAC) 11 conjugation of stereoprobe-labeled proteins to an azide-rhodamine reporter tag and in-gel fluorescence scanning, revealed that 36A-D displayed much lower overall proteomic reactivity compared to MY-11A/B//12A/B (Figure ). Several stereoselective protein interactions were also observed for the 3-amino stereoprobes at both test concentrations and time points of incubation (marked with red asterisks for the 20 µM and 50 μM, 3 h condition in Figure ). Based on these initial gel-ABPP data, we selected concentrations of 10 µM and 20 μM, re-spectively, to evaluate alkyne and competitor 3-amino stereoprobes by protein-directed mass spectrometry (MS)-ABPP. We performed protein-directed ABPP experiments by treating Ramos cells with non-alkyne competitor stereoprobes (20 μM; 37A-D) or dimethyl sulfoxide (DMSO) for 2 h, followed by treatment with the corresponding alkyne stereoprobes (10 μM; 36A-D) for 1 h. Cells were then lysed and stereoprobe-reactive proteins conjugated to biotin-azide by CuAAC, isolated with streptavidin beads, digested with trypsin, labelled with tandem mass tags (TMT), and identified (MS1/MS2 analysis) and quantified (MS3 analysis) by multiplexed (TMT16plex) MS-based proteomics (Figure ). Proteins showing greater than threefold enrichment by one stereoprobe compared to its enantiomer were assigned as stereoselectively enriched proteins. These proteins were further designated as being stereoselectively liganded if pretreatment by the corresponding competitor probe resulted in >50% blockade of stereoselective enrichment as compared to pretreatment with DMSO.