Complete de novo Sequencing of Circulating Antibodies by Top-Down Proteomics – AntiboTOP

Therapeutic antibodies (Abs) are a very potent class of newly developed and approved drugs. The discovery and development of these large-size biologics require the characterization of endogenous Abs, which remains a difficult and tedious task. This key step includes the identification of the Abs’ amino acid sequence and all post-translational modifications (PTMs). The commonly employed DNA/RNA sequencing methods present significant limitations and provide knowledge only at the genomic level, which precludes information on modifications of the mature and functional protein forms of Abs (proteoforms).
Mass spectrometry (MS) is an alternative tool for obtaining information directly at the relevant protein level. Bottom-up proteomics (BUP), which is based on the LC-MS/MS of peptides generated by the enzymatic digestion of Abs, is the primary line of their analysis nowadays. BUP can efficiently inform on both sequence and PTMs, but it lacks proteoform-level specificity. Alternative and complementary MS approaches have thus emerged, either at the intact Ab (top-down proteomics, TDP) or subunit (middle-down proteomics) level. However, none has proved efficient enough to allow endogenous Ab proteoforms to be fully characterized. In addition, although de novo sequencing tools exist for peptides, there is no such tool at the proteoform level.
The goal of the AntiboTOP project is to achieve the complete de novo sequencing of specific circulating Abs using innovative TDP approaches including a novel data processing and analysis pipeline. Two unique in France MS platforms will be made available for the AntiboTOP project: a Q-Exactive HF Orbitrap equipped with an Omnitrap and the Orbitrap Tribrid Eclipse, which is currently the most powerful commercial platform for TDP. The Omnitrap is an original device offering an arsenal of ion activation-dissociation techniques. The Omnitrap-Orbitrap system is equipped with a high-performance data acquisition system, FTMS Booster, giving access to the unreduced raw data (time-domain transients), and thus maximizing the extraction of information from the experimental set-ups. To enhance the performance of the Eclipse platform, we plan to equip it with the latter capability too.
The project is divided in three work packages. WP1 is dedicated to the experimental optimization of all LC-MS/MS parameters (chromatographic separation, fragmentation type,…), the evaluation of the requirements for data processing/analysis, as well as the capabilities of the existing software tools. WP1 will be performed on a simple mixture of well-characterized mAbs. In WP2, we will develop innovative and implement the most promising data processing/analysis methods to optimize the quality and confidence of the information extracted from the LC-MS/MS runs. The first part of the software will be dedicated to targeted TDP experiments (proteoform confirmation) and further extended to untargeted experiments (proteoform discovery), and, finally, to de novo sequencing. For the latter, we will integrate other sources of Abs information (e.g. sequence constraints). Finally, in WP3, we will use the optimized workflows developed in WP1 and WP2 for the analysis of circulating Abs immunopurified from the sera of patients either vaccinated or who recovered from Covid-19.
AntiboTOP is a multi-disciplinary project that relies on the unique complementarity and expertise of the MSBio Unit (J. Chamot-Rooke) at Institut Pasteur (biology, experimental MS) and a small deep-tech company Spectrotech SAS (Lyon) led by Y. Tsybin (physics, data processing and analysis algorithms). AntiboTOP will make, for the first time, the de novo sequencing of endogenous Abs by MS feasible. This represents a breakthrough in the field of immunology with a significant potential economic and industrial impact. The developed solutions will be aimed at wide dissemination and commercialization among academic and industrial analytical laboratories.