Having established the Trx1 kinetic trapping approach for soluble target proteins, we asked whether the kinetic trapping technique can also be applied to the surface of intact cells in culture. Given previous indications of disulfide bond exchange between CD4 and wild-type Trx1 (Matthias et al, 2002) , we asked whether kinetic trapping would enable us to detect this interaction on the surface of the CD4 positive promyelocytic cell line U937. In brief, we allowed mutant Trx1 to interact with the surface of live cells, removed unreacted oxidoreductase by washing and collected disulfide-linked Trx1 complexes from cellular lysates by and non-trapping (SSAAA) mutants of Trx1 were incubated with a o30 kDa fraction prepared from fresh human serum. Disulfide-linked complexes were analyzed by colloidal Coomassie staining under non-reducing and reducing conditions. The Trx1-Prx2 conjugate and the Trx1 dimer as well as monomeric Prx2 and Trx1 are indicated. (E) Kinetic trapping is mediated by specific protein-protein interactions. Cytosolic proteins from digitonin-permeabilized Jurkat cells were incubated with SAv sepharose, Grx1(CSAAA) or Trx1(CSAAA). Disulfide-linked complexes were analyzed by silver staining under nonreducing and reducing conditions. Trx1 conjugates formed with Prx1 and Prx2 are indicated. Other bands correspond to additional cytosolic proteins interacting with Trx1. streptavidin (SAv) affinity purification. We found that cell surface CD4 forms a mixed disulfide with exogenously added Trx1(CSAAA) (Supplementary Figure S1 , left panel), which could be dissociated by DTT treatment (Supplementary Figure S1 , right panel). This result confirmed that kinetic trapping can indeed be used to identify specific Trx1-reactive cell surface proteins and should therefore allow de novo identification of previously unknown cell surface target proteins.