LXJ Poster Note PN-28 A Fully Automated Robotic Set-Up based on Ultrahigh-Resolution QTOF Mass Spectrometry for Hydrogen Exchange Experiments Introduction The activity of most therapeutic antibodies is directed by their ability to link to a target antigen. Localizing the amino acids (AAs) responsible for this interaction is mandatory from a mechanistic point of view, but also for intellectual property protection. Epitope mapping is performed with an array of techniques, including hydrogen exchange mass spectrometry (HX-MS). With HX-MS solvent accessibility of the polypeptide backbone is measured and related to the proteins secondary structureas well as monitoring conformational changes occurring during antigen binding. While HX-MS experiments provide valuable information which is difficult to obtain by alternative techniques, they are also demanding in several aspects. On one hand they require exact timing and reproducibility in sample preparation. On the other hand full sensitivity at high mass resolution and isotopic fidelity is necessary for data interpretation. Here we established a system to meet these requirements (Fig. 1) and studied its performance using a biologically relevant model system. Authors J2S J. Wiesner, N. Hentze, M. P. Mayer ZMBH Uni. Heidelberg, Heidelberg, Germany E. Belau, W. Jabs Bruker Daltonik GmbH, Bremen, Germany C. Evans Bruker Ltd., Coventry, UK G. Tremintin Bruker Daltonics Inc., Fremont, CA, USA Keywords Instrumentation and Software Biopharma UHR-QTOF Hydrogen Exchange (HX) maXis II Protein Structure studies HDX system Higher Order Structure impact II Protein-Protein Interactions

Methods • Instrumental Setup: Liquid handling robot (HDX PAL3, LEAP Technologies), UHPLC-system (1290 Infinity II, Agilent) and a ultra-high resolution (UHR) QTOF mass spectrometer (maXis, Bruker). • Benchmark system used as model for detecting conformational changes caused by binding: E. coli Hsp90 (HtpG), a 140 kDa dimer, which undergoes significant conformational changes in response to ATP binding [Graf et al. EMBO J. 28, 602-613 (2009)]. Sophisticated liquid handling robot specialized for HX-MS experiments • HX-MS Experiment: HtpG and HtpG+ATP samples were prepared at 5 different labeling...

• S/MS experiments of non-deuterated HtpG after M pepsin digestion yielded 179 identified peptides with 97% sequence coverage. After manual evaluation and curation of the data, the list was reduced to 47 peptides representing 95% sequence coverage. These 47 peptides were used for the subsequent data analysis (Fig. 4). • fully automated robotic setup based on UHR-QTOF MS A was established providing the following advantages for HX-MS: • TP binding induced pronounced conformational changes, A resulting in a slow exchanging tensed state (Fig. 4 and Fig. 5), with the highest degree of HX protection...

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