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Use of tandem Biacore–mass spectrometry to identify platelet membrane targets of novel monoclonal antibodies by Catherine Ravanat; Virginie Wurtz; Philippe Ohlmann; Marie Fichter; Jean-Pierre Cazenave; Alain VanDorsselaer; François Lanza; Christian Gachet is a scholarly article available to read on EtoBox.

What is Use of tandem Biacore–mass spectrometry to identify platelet membrane targets of novel monoclonal antibodies about?

The monoclonal antibodies (mAbs) ALMA.17 and ALMA.7 recognize human platelet membrane proteins. ALMA.17 is directed against alpha(IIb)beta(3) integrin, but the target of ALMA.7 was unknown previously. Tandem Biacore micropurification and mass spectrometry (MS) analysis of a platelet membrane lysate was used to identify the target of ALMA.7. Detergent lysates enriched in membrane proteins were perfused over immobilized ALMA.17 or ALMA.7 in a Biacore system. The captured proteins were eluted, concentrated on C3 magnetic beads, and digested with trypsin before nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Critical adjustments needed to be made in (i) the detergent mixture to preserve protein antigenicity and sensor chip integrity and (ii) the method of trypsin digestion to concentrate the proteins and use elution buffers that do not interfere with MS. The target of ALMA.17 was confirmed to be alpha(IIb)beta(3) integrin, whereas that of ALMA.7 was identified as CD226 (PTA-1, DNAM-1, TLiSa-1). This was confirmed by immunoassays comparing ALMA.7 with a commercial anti-CD226 mAb. Thus, a tandem Biacore and nano LC-MS/MS strategy allowed unambiguous identificatio

Author

Catherine Ravanat; Virginie Wurtz; Philippe Ohlmann; Marie Fichter; Jean-Pierre Cazenave; Alain VanDorsselaer; François Lanza; Christian Gachet

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2009

Language

EN