Fig. 1: Identification of Kmeas on CypA protein.

a Tandem mass spectrum of BVSB mediated cross-linked peptide between TXN1 and CypA. b Previously, protein interaction of TXN1 and CypA was identified in TXN1 His-tag pull down³⁸. To validate protein interaction of TXN1 and CypA, CypA IP experiment was performed and TXN1 was identified in CypA IP samples. c, d HEK293T cells were transfected with CpyA-Strep, Strep-tag purified CypA was digested with trypsin. Digested peptides were analyzed by mass spectrometry. Tandem mass spectrum of peptides contained methacrylation on CypA Lys131 (c) and Lys125 (d). The K in red represents the Kmea modification. e Validation of Kmea on CypA by Western blot analysis. When CypA Lys125 was mutated to Arg, the Kmea level of CypA dramatically decreased. The experiment was repeated three times with similar results. When CypA Lys131 was mutated to Arg, the Kmea level of CypA partially decreased. f Proposed fragmentation pathway of CycIm ion from methacrylated peptide. g CycIm ion was mapped on MS/MS spectrum of methacrylated peptide (Kmea on CypA Lys131). h Confirm CycIm ion with chemically synthesized standard methacrylated peptide.