Integrated tools for higher order structure determination by cross-link analysis

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Summary The use of mass spectrometry (MS) for quantitative protein characterization has increased dramatically; identifying and quantifying thousands of proteins is no longer a heroic task nor is characterizing therapeutic proteins in detail. Due to widely available software, quantitative mass spectrometry has become routine and widely used for addressing questions in basic biology, translational medicine and drug development. MS Methods are now poised to have similar impact on the study of protein-protein interaction and higher order structure (HOS) determination. In particular, crosslinking mass spectrometry, which obtains distance and accessibility constraints from two-ended chemical modifications, is emerging as a powerful tool to map protein-protein interaction interfaces. Recent years have seen large improvements in crosslink mass spectrometry. Yet despite these advances, crosslink analysis is not yet a technique that has found widespread use, due to a range of problems such as the low abundance of crosslinked peptides, unavailability of proprietary reagents, the need for different search algorithms adopted to specific cross-linkers, and the difficulty of reliably characterizing crosslinked peptide spectra. To alleviate these problems and democratize the use of crosslinking, we propose to develop standardized and effective methods for crosslinking in conjunction with sensitive and accurate search algorithms, incorporated into our well-established Byonic search engine. Our algorithms will support almost all commercially available crosslinking chemistries and most major peptide fragmentation methods (ETD, CAD/HCD, EthcD), as well as cleavable and non-cleavable crosslinkers. The near universal applicability and ease-of-use of the software will promote rapid adoption of crosslinking mass spectrometry in academic research and biopharmaceutical development.