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., Curnis, F., Marazzini, L., Colombo, R., Sacchi, A., Corti, A., et al. (2001). Biotinylation sites of tumor necrosis factor-alpha determined by liquid chromatography-mass spectrometry. ANALYTICAL BIOCHEMISTRY, 298(2), 181-188 [10.1006/abio.2001.5374].

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

MAGNI, FULVIO;COLOMBO, ROBERTO;KIENLE, MARZIA DONATELLA
2001

Abstract

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.
Articolo in rivista - Articolo scientifico
tumor necrosis factor; mass spectrometry; biotin; tumor therapy; electrospray; HPLC
English
181
188
8
Magni, F., Curnis, F., Marazzini, L., Colombo, R., Sacchi, A., Corti, A., et al. (2001). Biotinylation sites of tumor necrosis factor-alpha determined by liquid chromatography-mass spectrometry. ANALYTICAL BIOCHEMISTRY, 298(2), 181-188 [10.1006/abio.2001.5374].
Magni, F; Curnis, F; Marazzini, L; Colombo, R; Sacchi, A; Corti, A; Kienle, M
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/15711
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