High-Throughput Characterization of Complex Crosslinked Proteins using Byonic and ByologicRyan D. Leib1; Chris Becker2; Yong Kil2; Pierre Allemand2, St John Skilton2; Eric Carlson2; Christopher M. Adams1; Allis Chien1
1Stanford University Mass Spectrometry, Stanford, CA 2Protein Metrics Inc.
Byologic® Features
Acknowledgment & ReferenceThis 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
Prot-2Prot-1
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 XIC9. 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
