Biotinylation sites of tumor necrosis factor-alpha determined by liquid chromatography-mass spectrometry

Tumor pretargeting with biotinylated antibody/avidin complexes improves the therapeutic index of systemically administered biotin-tumor necrosis facto r (TNF) conjugates. Since the number of biotins in this conjugate is known to be critical for activity, we have characterized the structure of differe nt biotin-TNF conjugates, prepared by reaction with D-biotinyl-6-aminocapro ic acid N-hydroxysuccinimide ester and identified the biotinylation sites b y trypsin digestion, reverse-phase chromatography, and electrospray mass sp ectrometry analyses. The results have shown that N-terminal valine is a pre ferential biotinylation site at pH 5.8, half of biotins being located on th e alpha -amino group of this residue in a conjugate bearing one biotin/trim er (on average). Moreover, evidence has been obtained to suggest that the r emaining part of biotins are linked to the E-amino group of lysine 128, 112 , and 65, while lysine 11, 90, and 98 were practically unmodified. No evide nce of O-biotinylation of serine, threonine and tyrosine was obtained. (C) 2001 Academic Press.