AlphaCross-XL: a seamless tool for automated and proteome-scale mapping of crosslinked peptides onto three-dimensional protein structures

Crosslinking mass spectrometry (XL-MS) is an exciting proteomics technology to capture native protein conformations in real time within biological systems. Historically, however, implementation of this technology has typically been limited to single purified recombinant proteins or in vitro assembled protein complexes. These limitations are associated with inherent challenges in XL-MS analysis, including extremely low abundance of crosslinked (XL) peptides and complex deconvolution of XL peptide-derived spectral data. However, impressive recent developments in computation and instrumentation have now made it feasible to address biological questions using proteome-wide XL-MS analysis. Although some XL mapping software tools exist, these require manual input of specific Protein Data Bank (PDB) structures at the single protein level, and do not function at the high throughput scale required to analyze datasets derived from thousands of proteins. To address this need, we therefore sought to develop a strategy enabling automated mapping of XL peptides onto the three-dimensional (3D) structures of proteins, at a proteome-wide scale. Herein we describe AlphaCross-XL, a first-in-class seamless computational tool for automated mapping of XL peptides onto the protein structures for intra-protein crosslinks and loop-links. The AlphaCross-XL software first retrieves protein structures from the AlphaFold Protein Structure Database and maps all the identified crosslinks onto the 3D structure. It also calculates the Euclidian distance between the crosslinked residues and reports the violated and satisfied crosslink distances based on a user-defined distance threshold, which is visually discriminated by color in PyMOL. Lastly, the tool also supports further validation of user-submitted protein structures, that can include any computer-predicted protein structure and experimentally derived protein structures (i.e., from PDB). AlphaCross-XL is available at https://github.com/sanjyotshenoy/alphacross-xl .