Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure.

Crosslinking mass spectrometry (XL-MS) has the potential to map the interactome of the cell with high resolution and depth of coverage. However, current in vivo XL-MS methods are hampered by crosslinkers that demonstrate low cell permeability and require long reaction times. Consequently, interactome sampling is not high and long incubation times can distort the cell, bringing into question the validity any protein interactions identified by the method. We address these issues with a fast formaldehyde-based fixation method applied prior to the introduction of secondary crosslinkers. Using human A549 cells and a range of reagents, we show that 4% formaldehyde fixation with membrane permeabilization preserves cellular ultrastructure and simultaneously improves reaction conditions for in situ XL-MS. Protein labeling yields can be increased even for nominally membrane-permeable reagents, and surprisingly, high-concentration formaldehyde does not compete with conventional amine-reactive crosslinking reagents. Prefixation with permeabilization uncouples cellular dynamics from crosslinker dynamics, enhancing control over crosslinking yield and permitting the use of any chemical crosslinker.

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA.

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology.

We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

High-Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media.

Affinity-purification mass spectrometry (AP-MS) is an established technique for identifying protein-protein interactions (PPIs). The basic technology involves immobilizing a high-specificity ligand to a solid-phase support (e.g., an agarose or magnetic bead) to pull down protein(s) of interest from cell lysates. Although these supports are engineered to minimize interactions with background protein, the conventional method recovers mostly nonspecific binders. The law of mass action for dilute solutions has taught us to use an excess of beads to capture all target proteins, especially weakly interacting ones. However, modern microbead technology presents a binding environment that is much different from a dilute solution. We describe a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing nonspecific binding. We demonstrate the concept with synthetic mixtures of tagged protein and illustrate performance with a variety of AP-MS experiment types. These include a BioID experiment targeting lamin-A interactors from HeLa cells and pulldowns using GFP-tagged proteins associated with a double-strand DNA repair mechanism. We show that efficient extraction requires saturation of the solid-phase support and that <10 nL of beads is sufficient to generate comprehensive protein interaction maps.

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity.

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

Mouse Model of Fragile X Syndrome Analyzed by Quantitative Proteomics: A Comparison of Methods.

Multiple methods for quantitative proteomics are available for proteome profiling. It is unclear which methods are most useful in situations involving deep proteome profiling and the detection of subtle distortions in the proteome. Here, we compared the performance of seven different strategies in the analysis of a mouse model of Fragile X Syndrome, involving the knockout of the fmr1 gene that is the leading cause of autism spectrum disorder. Focusing on the cerebellum, we show that data-independent acquisition (DIA) and the tandem mass tag (TMT)-based real-time search method (RTS) generated the most informative profiles, generating 334 and 329 significantly altered proteins, respectively, although the latter still suffered from ratio compression. Label-free methods such as BoxCar and a conventional data-dependent acquisition were too noisy to generate a reliable profile, while TMT methods that do not invoke RTS showed a suppressed dynamic range. The TMT method using the TMTpro reagents together with complementary ion quantification (ProC) overcomes ratio compression, but current limitations in ion detection reduce sensitivity. Overall, both DIA and RTS uncovered known regulators of the syndrome and detected alterations in calcium signaling pathways that are consistent with calcium deregulation recently observed in imaging studies. Data are available via ProteomeXchange with the identifier PXD039885.

High-Sensitivity Proteome-Scale Searches for Crosslinked Peptides Using CRIMP 2.0.

Crosslinking mass spectrometry (XL-MS) is a valuable technique for generating point-to-point distance measurements in protein space. However, cell-based XL-MS experiments require efficient software that can detect crosslinked peptides with sensitivity and controlled error rates. Many algorithms implement a filtering strategy designed to reduce the size of the database prior to mounting a search for crosslinks, but concern has been expressed over the possibility of reduced sensitivity using these strategies. We present a new scoring method that uses a rapid presearch method and a concept inspired by computer vision algorithms to resolve crosslinks from other conflicting reaction products. Searches of several curated crosslink datasets demonstrate high crosslink detection rates, and even the most complex proteome-level searches (using cleavable or noncleavable crosslinkers) can be completed efficiently on a conventional desktop computer. The detection of protein-protein interactions is increased twofold through the inclusion of compositional terms in the scoring equation. The combined functionality is made available as CRIMP 2.0 in the Mass Spec Studio.

13th annual symposium of the Canadian National Proteomics Network.

The 13th annual Canadian National Proteomics Network was held in May 2022 in Montreal, Quebec, Canada. More than 175 individuals participated in this dynamic and productive meeting either in-person or virtually. A pre-symposium organized by trainees and dedicated to highlighting the best and brightest emerging talent in proteomics across Canada preceded the main symposium, which welcomed plenary and invited speakers from around the world. The presentations covering ground-breaking science were interspersed with critical discussions on improving equity, diversity, and inclusion within the proteomics community across Canada, along with important networking opportunities for early-career researchers.

A Crosslinking Mass Spectrometry Protocol for the Structural Analysis of Microtubule-Associated Proteins.

Microtubule-associated proteins (MAPs) engage microtubules (MTs) to regulate both the MT state and wide variety of cytoskeletal functions. A comprehensive understanding of MAPs function requires the structural characterization of physical contacts MAPs make with other proteins, particularly when engaged with the microtubule (MT) lattice. Most of the interaction between MAPs and MTs evade classical structural determination techniques, as the interactions can be both heterogenous and sub-stoichiometric. Crosslinking mass spectrometry (XL-MS) can aid in MAP-MT structure analysis by providing a wealth of residue-based distance restraints. This protocol provides an XL-MS workflow for accurate and unbiased sampling of an equilibrated MAP-MT interaction, involving modifications to the preparation and validation of a MAP-MT construct suitable for crosslinking with fast-sampling heterobifunctional crosslinkers. The distance restrains obtained by this protocol can be used to generate accurate models assembled with an integrative structural modeling approach.

Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C-terminal domain.

Doublecortin (DCX) is a microtubule (MT)-associated protein that regulates MT structure and function during neuronal development and mutations in DCX lead to a spectrum of neurological disorders. The structural properties of MT-bound DCX that explain these disorders are incompletely determined. Here, we describe the molecular architecture of the DCX-MT complex through an integrative modeling approach that combines data from X-ray crystallography, cryo-electron microscopy, and a high-fidelity chemical crosslinking method. We demonstrate that DCX interacts with MTs through its N-terminal domain and induces a lattice-dependent self-association involving the C-terminal structured domain and its disordered tail, in a conformation that favors an open, domain-swapped state. The networked state can accommodate multiple different attachment points on the MT lattice, all of which orient the C-terminal tails away from the lattice. As numerous disease mutations cluster in the C-terminus, and regulatory phosphorylations cluster in its tail, our study shows that lattice-driven self-assembly is an important property of DCX.

IMProv: A Resource for Cross-link-Driven Structure Modeling that Accommodates Protein Dynamics.

Proteomics methodology has expanded to include protein structural analysis, primarily through cross-linking mass spectrometry (XL-MS) and hydrogen-deuterium exchange mass spectrometry (HX-MS). However, while the structural proteomics community has effective tools for primary data analysis, there is a need for structure modeling pipelines that are accessible to the proteomics specialist. Integrative structural biology requires the aggregation of multiple distinct types of data to generate models that satisfy all inputs. Here, we describe IMProv, an app in the Mass Spec Studio that combines XL-MS data with other structural data, such as cryo-EM densities and crystallographic structures, for integrative structure modeling on high-performance computing platforms. The resource provides an easily deployed bundle that includes the open-source Integrative Modeling Platform program (IMP) and its dependencies. IMProv also provides functionality to adjust cross-link distance restraints according to the underlying dynamics of cross-linked sites, as characterized by HX-MS. A dynamics-driven conditioning of restraint values can improve structure modeling precision, as illustrated by an integrative structure of the five-membered Polycomb Repressive Complex 2. IMProv is extensible to additional types of data.

Improving Spectral Validation Rates in Hydrogen-Deuterium Exchange Data Analysis.

The data analysis practices associated with hydrogen-deuterium exchange mass spectrometry (HX-MS) lag far behind that of most other MS-based protein analysis tools. A reliance on external tools from other fields and a persistent need for manual data validation restrict this powerful technology to the expert user. Here, we provide an extensive upgrade to the HX data analysis suite available in the Mass Spec Studio in the form of two new apps (HX-PIPE and HX-DEAL), completing a workflow that provides an HX-tailored peptide identification capability, accelerated validation routines, automated spectral deconvolution strategies, and a rich set of exportable graphics and statistical reports. With these new tools, we demonstrate that the peptide identifications obtained from undeuterated samples generated at the start of a project contain information that helps predict and control the extent of manual validation required. We also uncover a large fraction of HX-usable peptides that remains unidentified in most experiments. We show that automated spectral deconvolution routines can identify exchange regimes in a project-wide manner, although they remain difficult to accurately assign in all scenarios. Taken together, these new tools provide a robust and complete solution suitable for the analysis of high-complexity HX-MS data.

The active DNA-PK holoenzyme occupies a tensed state in a staggered synaptic complex.

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.

The substrate specificity of the human TRAPPII complex's Rab-guanine nucleotide exchange factor activity.

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

A substrate binding model for the KEOPS tRNA modifying complex.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

Harmonizing structural mass spectrometry analyses in the mass spec studio.

Structural Mass Spectrometry (SMS) provides a comprehensive toolbox for the analysis of protein structure and function. It offers multiple sources of structural information that are increasingly useful for integrative structural modeling of complex protein systems. As MS-based structural workflows scale to larger systems, consistent and coherent data interpretation resources are needed to better support modeling. Unlike the proteomics community, practitioners of SMS lack adequate computational tools. Here, we review new developments in the Mass Spec Studio: an expandable ecosystem of workflows for the analysis of complementary SMS techniques with linkages to modeling. Current functionality in the Studio (version 2) supports three major SMS workflows (crosslinking, hydrogen/deuterium exchange and covalent labelling) and two pipelines for structural modeling, with a special focus on data integration. The Mass Spec Studio is an architecture focused on rapid and robust extension of functionality by a community of developers. SIGNIFICANCE: This review surveys the new data analysis capabilities within the Mass Spec Studio, a rich framework for rapid software development specifically targeting the community of structural proteomics and structural mass spectrometry. Updates to crosslinking, hydrogen/deuterium-exchange and covalent labeling apps are provided as well as a utility for translating such analyses into restraints that support integrative structural modeling. These new capabilities, together with the underlying design tools and content, provide the community with a wealth of resources to tackle complex structural problem and design new approaches to data analysis.

Correlation between Labeling Yield and Surface Accessibility in Covalent Labeling Mass Spectrometry.

The functional properties of a protein are strongly influenced by its topography, or the solvent-facing contour map of its surface. Together with crosslinking, covalent labeling mass spectrometry (CL-MS) has the potential to contribute topographical data through the measurement of surface accessibility. However, recent efforts to correlate measures of surface accessibility with labeling yield have been met with mixed success. Most applications of CL-MS involve differential analysis of protein interactions (i.e., footprinting experiments) where such inconsistencies have limited effect. Extending CL-MS into structural analysis requires an improved evaluation of the relationship between labeling and surface exposure. In this study, we applied recently developed diazirine reagents to obtain deep coverage of the large motor domain of Eg5 (a mitotic kinesin), and together with computational methods we correlated labeling yields with accessibility data in a number of ways. We observe that correlations can indeed be seen at a local structural level, but these correlations do not extend across the structure. The lack of correlation arises from the influence of protein dynamics and chemical composition on reagent partitioning and, thus, also on labeling yield. We conclude that our use of CL-MS data should be considered in light of "chemical accessibility" rather than "solvent accessibility" and suggest that CL-MS data would be a useful tool in the fundamental study of protein-solute interactions.

Federating Structural Models and Data: Outcomes from A Workshop on Archiving Integrative Structures.

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.