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High-Throughput Characterization of Complex Crosslinked Proteins using Byonic and Byologic Ryan D. Leib 1 ; Chris Becker 2 ; Yong Kil 2 ; Pierre Allemand 2 , St John Skilton 2 ; Eric Carlson 2 ; Christopher M. Adams 1 ; Allis Chien 1 1 Stanford University Mass Spectrometry, Stanford, CA 2 Protein Metrics Inc. Acknowledgment & Reference This work was supported by NIH grant GM100634. 1. Komolov et al. Cell 2017, 169, 407-421 Byologic ® Features Using this workflow, we can readily analyze thousands of possible crosslinked peptides across diverse experiments. The goal is to identify the best crosslinks first as anchor structural constraints taking advantage of various linker types and proteolytic enzymes. This Byologic strategy can produce a robust candidate report in hours instead of days to weeks, and efficiently informs structural modeling efforts and biochemical experiments. Here, we identified the binding interface of b2AR-GRK5 using this method. Our crosslink predictions observed an ionic lock region opening and a rotation of the RH bundle. These experimental observations were further supported by complementary HDX, EM, and mutant crosslink analyses. This approach is scalable to other protein-protein interactions, including determination of complex stoichiometry and polymerization. Contact: [email protected] Byologic Features Methods Crosslink Data Analysis in Ten Easy Steps Xlinks Used to Define Structural Constraints Discussion and Conclusions New mass spectrometry methods to investigate protein-protein complexes, binding interfaces, and biopolymer oligomerization show tremendous promise as structural biology tools. In practice, improved data analysis tools and methods are needed to validate the rich but incredibly complex MS/MS fragmentation data. Here, we present a novel analysis approach for chemical crosslinking using features of Byonic and Byologic with an empirical grading system to quickly and robustly characterize a model G-protein-coupled receptor, b2 Adrenegic Receptor (b2AR) with its kinase (GRK5). While the structure of the individual proteins are determined, the molecular architecture of the docked interaction is poorly understood, and has important implications for a host of signaling pathways and protein recruitment mechanisms. MS2 Identifications Methods Project Window Protein Coverage XICs Peptide Windows Unreliable Chromatography Co-isolated Precursors MS/MS Alone Looks Reasonable… Summary tables take the output of this analysis to efficiently parse True Positives, False Positives, and Uncertains for group review, assignment, and reporting A parallel strategy using multiple crosslinkers, proteolytic enzymes, and fragmentation methods (ETD, HCD) was used to extensively characterize in vitro crosslinked GRK5-b2AR. Raw data were searched against a focused database in Byonic to identify thousands of candidate crosslinked peptides for review. Byologic was used to condense these many parallel experiments and facilitate rapid cross-link validation using an empirical rule strategy described here. This approach not only provides significant time savings over individual review of the independent output files, it provides more robust cross comparison between related experiments, and a robust permanent report generation for easy information transfer between researchers. Introduction Report 1. Group Related Experiments 2. Remove Unlinked Peptides 3. Identify Xlinks with Multiple Observations 4. Note Source Experiment of Peptide 5. Sequester Xlinks with Short Peptides 6. Verify MS/MS 7. Qualify MS1 Isolation 8. Validate XIC 9. Compare with Unlinked or in silico peptides 10. Record Validation Byologic Dashboard MS2 annotation and m/z errors Rapid Classification Direct Report Generation b2AR-GRK5 Crosslink Report Xlinks are categorized into true-positives, uncertain, and false-positives using this empirical workflow. “True positives” succeed on all rules, while “False Positives” fail least three of these rules. This strategy focuses on identifying the most likely xlinks first, and then can recover uncertain peptides using an easy, transferable, and repeatable framework for analysis. True positives anchor the b2AR-GRK5 structural determination, with uncertain assignments filling in based on 3D structural limitations. MS1 isotopes BUT 1. Docking of crystal structure 2. Opening of ionic lock region 3. Rotation of RH bundle
