Bacterial type III secretion systems are able to secrete membrane proteins. In this project we could show that cognate T3SS chaperones are able to bind to the transmembrane segments of T3SS substrates to avoid erroneous targeting to the bacterial inner membrane. Here, we studied the Salmonella SPI-2 T3SS secreted protein SseF and the interaction with its chaperone SscB by in vivo photocrosslinking. LC-MS/MS analysis was done to provide additional evidence that SscB is the interaction partner leading to the upshift of the SseF band in an SDS-PAGE analysis.