Fig. 4. Cysteine-to-lysine transfer (CLT) strategy with MESNa thioester 3: (a) general scheme, (b) LC-MS after reaction of native Fab with thioester 3, (c) LC-MS after transthioesterification of reduced Fab with thioester 3, (d) LC-MS of CLT conjugate 5, (e) LC-MS of reduced CLT conjugate 5, in which the cysteines have been capped with N-methylmaleimide (the masses include the expected addition of one N-methylmaleimide on the light chain and one on the heavy chain, see Fig. S15 for details†), (f) SDS-PAGE analysis: (M) molecular marker, (1) native Fab, (2) reduced Fab after transthioesterification with MESNa thioester 3, (3) acyl transfer (24 h), (4) acyl transfer (48 h), (5) acyl transfer (72 h), i.e. CLT conjugate 5 (see Fig. S17† for complete SDS-PAGE analysis); 0, 1, 2 refer to the number of acyl groups added per species.