Cysteine-directed PEGylation of FVIII is highly specific. (A) FVIII K1804C before (lanes 1,3) and after (lanes 2,4) conjugation with a 60-kDa PEG was separated on SDS-PAGE and stained with Coomassie Blue for protein (lanes 1,2) or with barium iodide for PEG (lanes 3 and 4). (B) FVIII K1804C before (lanes 1,3) and after (lanes 2,4) conjugation with a 60-kDa PEG was digested with thrombin, separated on SDS-PAGE, and stained with Coomassie Blue for protein (lanes 1,2) or with barium iodide for PEG (lanes 3,4). (C-D) FVIII F129C before (C) and after (D) conjugation with a 60-kDa PEG was digested with thrombin and analyzed by MALDI-MS. Samples were spotted onto a MALDI target (0.3 μL) with the same volume of a MALDI matrix composed of sinappinic acid (Sigma-Aldrich) in 0.1% trifluoroacetic acid in water (70%) and acetonitrile (30%). HC indicates heavy chain; and LC, light chain.