Camille Garcia (Paris / FR), Konstantin Nagornov (Lausanne / CH), Athanasios Smyrnakis (Athens / GR), Tingting Fu (Paris / FR), Anton Kozhinov (Lausanne / CH), Dimitrios Papanastasiou (Athens / GR), Yury Tsybin (Lausanne / CH), Julia Chamot-Rooke (Paris / FR)
A novel instrumental platform called Omnitrap-Orbitrap-Booster with enhanced high-resolution top-down proteomics capabilities was recently introduced by our team. This platform is composed of a Q-Exactive HF Orbitrap interfaced with a multimodal MS/MS Omnitrap, offering ECD and CID, and an external high-performance data acquisition and processing system, FTMS Booster X2. The latter allows to record time-domain transients of any length. Utilizing optimized experimental parameters and refined data processing, we show that this platform outperforms state-of-the-art instruments and allows the complete sequencing of a clinical antibody light chain on an LC time scale. Our platform's efficacy is also showcased through the analysis of other antibody subunits including mixtures.
LC-MS/MS experiments were performed on the Omnitrap-Orbitrap-Booster platform (ECD, CID) as well as on an Eclipse Tribrid Orbitrap (EThcD) for comparison. Samples included an antibody light chain extracted from the urine of a multiple myeloma patient as well as commercial mAbs digested into various subunits. The time-domain transients collected by the FTMS Booster X2 (Spectroswiss) were converted into absorption mode FT (aFT). Conventional eFT mass spectra (.RAW) were collected in parallel. Data were analyzed with Proteome Discoverer, Peak-by-Peak (Spectroswiss), and in-house developed software tools.
The experimental parameters of the Omnitrap-Orbitrap-Booster platform were first optimized, and we found ECD (100 ms) to lead to the best sequence coverage for the LC-MS/MS analysis of our reduced/alkylated clinical light chain (25 kDa). The analysis of data acquired from the fragmentation of a single charge state (23+) demonstrated a sequence coverage increase of about 10% for aFT data in comparison with the eFT mass spectra acquired in parallel. Averaging time-domain data across five technical replicates further improved sequence coverage but only by a few % to reach 91% sequence coverage for the same sample.
We then acquired LC-MS/MS ECD datasets for charge states 15-25 with a single charge state resolution. Combining these results provided a sequence coverage of 100% already from the eFT mass spectra at 3 ppm mass accuracy. The time-domain data averaging produced additional benefits including enhanced confidence in product ion assignment. Our data acquisition strategy was optimized to allow the sequential fragmentation of multiple charge states in a single LC-MS/MS run. This approach circumvents the problem of overlap between charge-reduced species and product ions. Our optimized pipeline is also now currently applied to the top-down analysis of other antibody subunits (25/50 kDa) previously studied with the most recent platforms. Our objective is to achieve a 100% sequence coverage in a minimum of LC-MS/MS runs, which paves the way to the analysis of antibody mixtures and opens the attractive possibility of de novo sequencing from top-down data.