To test whether Trx1(C35S)-based trapping is capable of identifying known Trx1 target proteins, Trx1(CSAAA) was allowed to react with cytosolic proteins released from digitoninpermeabilized cells. Incubation led to the formation of a reproducible pattern of distinct mixed disulfide conjugates as visualized by silver staining of a SDS-PAGE gel under nonreducing conditions ( Figure 1C , lane 7). In accordance with the trapping mechanism, conjugation strictly depended on the availability of the N-terminal thiol (Cys-32) and the concurrent absence of the C-terminal thiol (Cys-35), as wild-type or cysteine-free Trx1 did not capture any proteins ( Figure 1C, lanes 3, 5 and 9 ). The pattern of trapped proteins was not significantly influenced by the presence or absence of the non-catalytic cysteines (data not shown). The main cytosolic interaction partner of Trx1(CSAAA) was identified as peroxiredoxin-1 (Prx1) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The Trx1-Prx1 association was further confirmed by immunoblotting (data not shown). The Trx1-Prx1 disulfide-linked conjugate is maintained under non-reducing conditions ( Figure 1C , lane 7) and cleaved into its monomer constituents under reducing conditions ( Figure 1C , lane 8).