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

Fulvio Magni, Flavio Curnis, Laura Marazzini, Roberto Colombo, Angelina Sacchi, Angelo Corti, Marzia Galli Kienle

Research output: Contribution to journalArticlepeer-review


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 α-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 ε-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.

Original languageEnglish
Pages (from-to)181-188
Number of pages8
JournalAnalytical Biochemistry
Issue number2
Publication statusPublished - Dec 15 2001


  • Biotin
  • Electrospray
  • HPLC
  • Mass spectrometry
  • Tumor necrosis factor
  • Tumor therapy

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology


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