While CETSA and GHR pulldown experiments allow ascertaining engagement of cellular SOCS2, distinguishing reversible binding from covalent binding remains a challenge. Towards evaluating fraction of SOCS2 covalently modified by MN714, we performed a SOCS2 immunoprecipitation (IP) in K562 cells treated with MN714, followed with tandem MS on the SOCS2 peptides (no significant cytotoxicity was observed with MN714 in K562 cells up to 3-10 μM, see Supplementary Figure ). Analysis of IP blots show that the anti-SOCS2 antibody recognizes both apo SOCS2 and MN551(unmasked MN714) modified SOCS2 efficiently (Figure ). Tandem MS analysis of the IP samples show that MN551 successfully modifies cellular SOCS2 and specifically targets Cys111 for reaction. Moreover, the MS2 spectra for cells treated with 1 μM and 10 μM MN714 show a dose dependent increase in levels of SOCS2 modification (Figure ). An analysis of SOCS2 peptides from cells treated with 10 μM MN714 show a complete saturation of Cys111 with no detectable levels of unmodified Cys111.