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 factor (TNF) conjugates. Since the number of biotins in this conjugate is known to be critical for activity, we have characterized the structure of different biotin-TNF conjugates, prepared by reaction with d-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester and identified the biotinylation sites by trypsin digestion, reverse-phase chromatography, and electrospray mass spectrometry analyses. The results have shown that N-terminal valine is a preferential biotinylation site at pH 5.8, half of biotins being located on the alpha-amino group of this residue in a conjugate bearing one biotin/trimer (on average). Moreover, evidence has been obtained to suggest that the remaining part of biotins are linked to the epsilon-amino group of lysine 128, 112, and 65, while lysine 11, 90, and 98 were practically unmodified. No evidence of O-biotinylation of serine, threonine and tyrosine was obtained.
Magni
F
F
Mass Spectrometry Laboratory, IRCCS S. Raffaele, Milan, Italy.
Curnis
F
F
Marazzini
L
L
Colombo
R
R
Sacchi
A
A
Corti
A
A
Kienle
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005347:Fibroblasts / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha; D001710:Biotin; D014357:Trypsin