Chemical cross‐linking and native mass spectrometry: A fruitful combination for structural biology

Multiprotein complexes are central to our understanding of cellular biology, as they play critical roles in nearly every biological process. Despite many impressive advances associated with structural characterization techniques, large and highly-dynamic protein complexes are too often refractory to analyze by conventional, high-resolution approaches. To fill this gap, ion mobility-mass spectrometry (IM-MS) methods have emerged as a promising approach for characterizing the structures of challenging assemblies due in large part to the ability of these methods to characterize the composition, connectivity, and topology of large, labile complexes. In this Critical Insight, we present a series of bioinformatics studies aimed at assessing the information content of IM-MS datasets for building models of multiprotein structure. Our computational data highlights the limits of current coarse-graining approaches, and compelled us to develop an improved workflow for multiprotein topology modeling, which we benchmark against a subset of the multiprotein complexes within the PDB. This improved workflow has allowed us to ascertain both the minimal experimental restraint sets required for generation of high-confidence multiprotein topologies, and quantify the ambiguity in models where insufficient IM-MS information is available. We conclude by projecting the future of IM-MS in the context of protein quaternary structure assignment, where we predict that a more complete knowledge of the ultimate information content and ambiguity within such models will undoubtedly lead to applications for a broader array of challenging biomolecular assemblies.

Section: Leveraging Symmetry and Modularity To Resolve Ambiguity Withmentioning

confidence: 99%

Coming to Grips with Ambiguity: Ion Mobility-Mass Spectrometry for Protein Quaternary Structure Assignment

Eschweiler

Frank

Ruotolo

2017

“…Hydrogen/deuterium exchange reports on secondary structure and dynamics [5][6][7][8]. Cross-linking provides distance constraints that can be essential for structure determination efforts [9][10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

Vahidi

Konermann

2016

Time (s)0Bleaching of the reporter dye cyanine-5 (Cy5) served as readout of the timedependent radical milieu. Surprisingly, Cy5 oxidation extends over tens of milliseconds. This time range is four orders of magnitude longer than expected from the FPOP literature. We demonstrate that the glutamine scavenger generates metastable secondary radicals in the FPOP solution, and that these radicals lengthen the time frame of Cy5 oxidation. Cy5 and similar dyes are widely used for monitoring the radical dose experienced by proteins in solution. The measured Cy5 kinetics thus strongly suggest that protein oxidation in FPOP extends over a much longer time window than previously thought (i.e., many milliseconds instead of one microsecond). The optical approach developed here should be suitable for assessing the performance of future FPOP-like techniques with improved temporal labeling characteristics.

“…In this approach, proteins and protein complexes are linked together using any of a myriad of available cross-linking reagents. The cross-linked proteins are then enzymatically digested to peptides and analyzed by LC-MS/MS, where cross-linked peptide sequences can be identified using specialized database search algorithms [4–5]. These cross-linked peptide spectrum matches (PSMs) contain two distinct peptides sequences that are used to infer proximal regions of protein structure from linearly distal domains, or domains from different proteins.…”

Section: Introductionmentioning

confidence: 99%

An Open Data Format for Visualization and Analysis of Cross-Linked Mass Spectrometry Results

Hoopmann

Mendoza

Deutsch

et al. 2016

Protein-protein interactions are an important element in the understanding of protein function, and chemical cross-linking shotgun mass spectrometry is rapidly becoming a routine approach to identify these specific interfaces and topographical interactions. Protein cross-link data analysis is aided by dozens of algorithm choices, but hindered by a lack of a common format for representing results. Consequently, interoperability between algorithms and pipelines utilizing chemical cross-linking remains a challenge. pepXML is an open, widely-used format for representing spectral search algorithm results that has facilitated information exchange and pipeline development for typical shotgun mass spectrometry analyses. We describe an extension of this format to incorporate cross-linking spectral search results. We demonstrate application of the extension by representing results of multiple cross-linking search algorithms. In addition, we demonstrate adapting existing pepXML-supporting software pipelines to analyze protein cross-linking results formatted in pepXML.