To identify the unknown Trx1 target protein, we performed cell surface trapping on a larger scale (5 Â10 9 LCL-721.220 cells), purified the Trx1-interacting surface protein by SAv affinity purification and visualized the protein by colloidal Coomassie staining ( Figure 3A , left panel). The 160 kDa band was absent in the control precipitation with Trx1(CCAAA). Corresponding bands from non-reducing and reducing lanes were subjected to tryptic digestion and LC-MS/MS analysis. From both samples the unknown protein was identified as TNFRSF8 (CD30), a member of the TNFR superfamily. To validate the direct covalent interaction between Trx1(CSAAA) and CD30, an aliquot of trapped complexes from the same experiment was separated under non-reducing and reducing conditions and subjected to immunoblotting analysis with anti-Trx1 ( Figure 3A , middle panel) and anti-CD30 antibodies ( Figure 3A , right panel), respectively. The observed mobility difference between non-reducing (NR) and reducing (R) lanes demonstrated the formation of a mixed disulfide conjugate ( Figure 3A , right panel). Additional immunoblotting experiments demonstrated that low nanomolar concentrations of Trx1(CSAAA) are sufficient to detect the interaction with CD30 ( Figure 3B ) and also confirmed that trapping of CD30 depends on the N-terminal cysteine of the CXXC motif ( Figure 3C, lanes 1-8) . Application of the Grx1 trapping mutant under the same conditions did not lead to its conjugation to CD30 ( Figure 