Real time optimization of electron-based fragmentation for middle and top-down proteomics in mass spectrometry

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The identification and quantification of biological macromolecules remains challenging despite major advances in the speed, resolution and mass accuracy of modern mass spectrometers. A key weakness with current instrumentation lies in the methods used to induce fragmentation. The reliance in particular on collision-induced dissociation (CID) has limited such analyses to bottom-up workflows of trypsin-digested peptides of 10-30 residues. At e-MSion, we have developed an efficient electron-fragmentation technology called ExD for large proteins and are now co-marketed our ExD Option with Agilent, and soon will be with Thermo and Waters instruments. What has really captured the interest of the biopharma and top-down communities in the past year is the exceptional sequence coverage of native proteins we obtain with the same ExD cell. The resulting spectra are less congested than those obtained with currently available ETD/UVPD/CID fragmentation methodologies. We have shown that our technology works faster and gives cleaner spectra with more complete dissociation with larger macromolecular protein complexes than has ever been possible before, while still preserving labile post translational modifications. In addition, fragmentation with higher energy electrons can be used to provide complementary data to improve protein and glycan identification. The challenge now has become how to optimally collect and process these data to maximize the utility of ExD fragmentation. Last summer, Xilinx released its Versal Adaptive Compute Acceleration Platform (ACAP), a massively parallel processor with 50 billion transistors targeted to transform digital signal processing, handling of big data and artificial intelligence. This ACAP technology has already accelerated Illumina DNA sequence assembly by 90-fold. Our feasibility question asks how to effectively harness this new highly parallelized technology to preprocess complex top-down mass spectra on- the-fly. This will allow us to actively optimize data acquisition by enabling adaptive operation of the ExD cell and mass spectrometer. The objective is to maximize both fragmentation and dissociation of native proteins, enabling faster and comprehensive characterization of challenging proteoforms important to the biopharmaceutical industry and biomedical researchers. Success will offer an extremely fast, cost-effective solution to characterize complexes of macromolecules under native conditions with increased accuracy, speed, and fewer misidentifications. Our ExD technology with the Versal ACAP can be both retrofitted into existing mass spectrometers as well as being available in new generations of mass spectrometers at a price below other less-effective alternative fragmentation technologies like ETD and UVPD. Thus, it will provide new abilities for many NIH investigators to advance basic research, probe disease mechanisms and permit more sophisticated searches for both diagnostic and therapeutic biomarkers.