Biotinylation Sites of Tumor Necrosis Factor- Determined by Liquid Chromatography–Mass Spectrometry Fulvio Magni,* Flavio Curnis,† Laura Marazzini,* Roberto Colombo,‡ Angelina Sacchi,† Angelo Corti,† and Marzia Galli Kienle‡ *Mass Spectrometry Laboratory, IRCCS S. Raffaele, Milan, Italy; †DIBIT, San Raffaele H Scientific Institute, Milan, Italy; ‡Department of Medical Chemistry and Biochemistry, University of Milan Bicocca, Milan, Italy; and §Catholic University Holy Heart of Milan, Milan, Italy Received February 5, 2001; published online October 18, 2001 Tumor pretargeting with biotinylated antibody/avi- din complexes improves the therapeutic index of sys- temically 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 -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 -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. © 2001 Academic Press Key Words: tumor necrosis factor; mass spectrome- try; biotin; tumor therapy; electrospray; HPLC. Human tumor necrosis factor  (TNF) 1 is a nonglyco homotrimeric protein produced by a large number of cell types and organs in response to various inflammatory and immunological stimuli. Several studies have shown that TNF is endowed with potent antitumor activity in animal models (1). However, the clinical use of TNF as an anticancer drug is limited by severe systemic tox- icity (2, 3). In the attempt to overcome this problem, we have recently developed a new strategy based on tumor pretargeting with biotinylated antibody/avidin com- plexes and systemic administration of biotinylated TNF (4, 5). This pretargeting approach was shown to increase at least five times the antitumor activity of biotinylated TNF in animal models, with no evidence of increased toxicity (6). Studies on the mechanism of action showed that biotin–TNF trimers can slowly dissociate from the targeted cells through trimer–monomer–trimer transi- tion and that the antitumor activity is based mainly on indirect effects, presumably on cells other than the tar- geted tumor cells (4, 6). We have also shown that nonbi- otinylated subunits must be present within TNF trimers for an efficient release of bioactive TNF from the target- ing complex. Since the number and the position of biotins in these conjugates is likely critical for the activity, the identification of the product present in the mixture after biotinylation is very useful. Mass spectrometry is a pow- erful tool to study covalent structure of proteins in con- junction with enzymatic digestion which provides short peptides to be used for additional information on the sequence (7–10). In this work we have characterized the structure of different biotin–TNF conjugates and identified the bi- otinylation sites, using total and partial trypsin diges- tion and mass spectrometry analysis. MATERIALS AND METHODS Preparation of TNF and Biotin–TNF Conjugates Human TNF (5  10 7 U/mg) was produced by recom- binant DNA technology. The cDNA coding for TNF (11) was cloned in pET-11b (Novagen, Madison, WI) and used to transform BL21 (DE3) Escherichia coli cells (Novagen). Soluble TNF was recovered from 2-liter cultures by bacterial sonication in 2 mM EDTA, 20 mM Tris–HCl, pH 8.0, followed by centrifugation (15,000g, 20 min, 4°C). The extract was mixed with ammonium sulfate (25% of saturation), left for 1 h at 4°C, and 1 Abbreviations used: TNF, tumor necrosis factor; TFA, trifluoro- acetic acid; ESI-MS, electrospray mass ionization; LC, liquid chro- matography. 0003-2697/01 $35.00 181 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 298, 181–188 (2001) doi:10.1006/abio.2001.5374, available online at http://www.idealibrary.com on