scholarly journals Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

2019 ◽  
Author(s):  
Tamar Tayri-Wilk ◽  
Moriya Slavin ◽  
Joanna Zamel ◽  
Ayelet Blass ◽  
Shon Cohen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Carbon Atom ◽  
Mass Spectrometry Data ◽  
Cross Linking ◽  
Atomic Structures ◽  
Cross Links ◽  
Structured Proteins ◽  
Structural Insights ◽  
Current Mechanism

AbstractFormaldehyde is a widely used fixative in biology and medicine. The current mechanism of formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this mechanism. Instead, the data reveal that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein βNAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.

scholarly journals Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

2021 ◽  
Author(s):  
Moriya Slavin ◽  
Joanna Zamel ◽  
Keren Zohar ◽  
Siona Eliyahu ◽  
Merav Braitbard ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Full Length ◽  
Integrative Model ◽  
Cross Linking ◽  
Cross Link ◽  
Integrative Modeling ◽  
Cellular Context ◽  
Cross Links ◽  
The Cross

AbstractAtomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

scholarly journals Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2

2021 ◽  
Vol 118 (34) ◽  
pp. e2103554118
Author(s):  
Moriya Slavin ◽  
Joanna Zamel ◽  
Keren Zohar ◽  
Tsiona Eliyahu ◽  
Merav Braitbard ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Full Length ◽  
Integrative Model ◽  
Cross Linking ◽  
Cross Link ◽  
Integrative Modeling ◽  
Cellular Context ◽  
Cross Links ◽  
The Cross

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication–transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

scholarly journals CLMSVault: A Software Suite for Protein Cross-Linking Mass-Spectrometry Data Analysis and Visualization

2017 ◽  
Vol 16 (7) ◽  
pp. 2645-2652 ◽  
Author(s):  
Mathieu Courcelles ◽  
Jasmin Coulombe-Huntington ◽  
Émilie Cossette ◽  
Anne-Claude Gingras ◽  
Pierre Thibault ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Data Analysis ◽  
Mass Spectrometry Data ◽  
Cross Linking ◽  
Software Suite ◽  
Protein Cross Linking

scholarly journals Cross-linking mass spectrometry reveals structural insights of the glutamine synthetase from Leishmania braziliensis

2021 ◽  
Vol 116 ◽  
Author(s):  
Jhenifer Yonara de Lima ◽  
Marlon Dias Mariano Santos ◽  
Mario Tyago Murakami ◽  
Paulo Costa Carvalho ◽  
Tatiana de Arruda Campos Brasil de Souza
Keyword(s):  
Mass Spectrometry ◽  
Glutamine Synthetase ◽  
Cross Linking ◽  
Leishmania Braziliensis ◽  
Structural Insights

Structural insights into calmodulin/Munc13 interaction

2014 ◽  
Vol 395 (7-8) ◽  
pp. 763-768 ◽  
Author(s):  
Sabine Herbst ◽  
Noa Lipstein ◽  
Olaf Jahn ◽  
Andrea Sinz
Keyword(s):  
Mass Spectrometry ◽  
Synaptic Plasticity ◽  
Binding Site ◽  
Cross Linking ◽  
Binding Motif ◽  
Binding Modes ◽  
Short Term ◽  
Common Domain ◽  
Structural Insights ◽  
Chemical Cross Linking

Abstract Munc13 proteins are essential presynaptic regulators that mediate synaptic vesicle priming and play a role in the regulation of neuronal short-term synaptic plasticity. All four Munc13 isoforms share a common domain structure, including a calmodulin (CaM) binding site in their otherwise divergent N-termini. Here, we summarize recent results on the investigation of the CaM/Munc13 interaction. By combining chemical cross-linking, photoaffinity labeling, and mass spectrometry, we showed that all neuronal Munc13 isoforms exhibit similar CaM binding modes. Moreover, we demonstrated that the 1-5-8-26 CaM binding motif discovered in Munc13-1 cannot be induced in the classical CaM target skMLCK, indicating unique features of the Munc13 CaM binding motif.

scholarly journals Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

2018 ◽  
Author(s):  
Therese Dau ◽  
Kapil Gupta ◽  
Imre Berger ◽  
Juri Rappsilber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Structures ◽  
Cross Linking ◽  
Cleavage Sites ◽  
Protein Modelling ◽  
Protein Protein Interactions ◽  
Important Approach ◽  
Cross Links ◽  
Sequential Digestion

ABSTRACTCross-linking/mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

scholarly journals Optimized fragmentation improves the identification of peptides cross-linked using MS-cleavable reagents

2018 ◽  
Author(s):  
Christian E. Stieger ◽  
Philipp Doppler ◽  
Karl Mechtler
Keyword(s):  
Mass Spectrometry ◽  
Quality Data ◽  
Cross Linking ◽  
High Complexity ◽  
Mass Spectrometers ◽  
Acquisition Strategies ◽  
Cross Links ◽  
The Cross ◽  
The Individual

ABSTRACTCross-linking mass spectrometry (XLMS) is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by non-specialist laboratories. Specifically, the use of novel mass spectrometry-cleavable (MS-cleavable) reagents dramatically reduces complexity of the data by providing i) characteristic reporter ions and ii) the mass of the individual peptides, rather than that of the cross-linked moiety. However, optimum acquisition strategies to obtain the best quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone is yet to be achieved. Therefore, we have carefully investigated and optimized MS parameters to facilitate the identification of disuccinimidyl sulfoxide (DSSO)- based cross-links on HCD-equipped mass spectrometers. From the comparison of 9 different fragmentation energies we chose several stepped-HCD fragmentation methods that were evaluated on a variety of cross-linked proteins. The optimal stepped-HCD-method was then directly compared with previously described methods using an Orbitrap Fusion™ Lumos™ TribridTM instrument using a high-complexity sample. The final results indicate that our stepped-HCD method is able to identify more cross-links than other methods, mitigating the need for multistage MS (MSn) enabled instrumentation and alternative dissociation techniques.

scholarly journals Mass Spectrometric Identification of a Novel Factor XIIIa Cross-Linking Site in Fibrinogen

Proteomes ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 43
Author(s):  
Mariya E. Semkova ◽  
J. Justin Hsuan
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Peptide Identification ◽  
Mass Spectrometric ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Spectrometric Analysis ◽  
Peptide Analysis ◽  
Linear Peptide ◽  
Cross Links

Transglutaminases are a class of enzymes that catalyze the formation of a protein:protein cross-link between a lysine and a glutamine residue. These cross-links play important roles in diverse biological processes. Analysis of cross-linking sites in target proteins is required to elucidate their molecular action on target protein function and the molecular specificity of different transglutaminase isozymes. Mass-spectrometry using settings designed for linear peptide analysis and software designed for the analysis of disulfide bridges and chemical cross-links have previously been employed to identify transglutaminase cross-linking sites in proteins. As no control peptide with which to assess and improve the mass spectrometric analysis of TG cross-linked proteins was available, we developed a method for the enzymatic synthesis of a well-defined transglutaminase cross-linked peptide pair that mimics a predicted tryptic digestion product of collagen I. We then used this model peptide to determine optimal score thresholds for correct peptide identification from y- and b-ion series of fragments produced by collision-induced dissociation. We employed these settings in an analysis of fibrinogen cross-linked by the transglutaminase Factor XIIIa. This approach resulted in identification of a novel cross-linked peptide in the gamma subunit. We discuss the difference in behavior of ions derived from different cross-linked peptide sequences and the consequent demand for a more tailored mass spectrometry approach for cross-linked peptide identification compared to that routinely used for linear peptide analysis.

scholarly journals The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

2020 ◽  
Author(s):  
Alan Rodriguez Carvajal ◽  
Carlos Gomez Diaz ◽  
Antonia Vogel ◽  
Adar Sonn-Segev ◽  
Katrin Schodl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Catalytic Activity ◽  
Ubiquitin Ligase ◽  
Bond Formation ◽  
Mass Spectrometry Data ◽  
Cross Linking ◽  
Concerted Action ◽  
Ubiquitin Chain ◽  
C Terminus

AbstractThe linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. We present the first 3D reconstruction of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains. Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching confirming its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.

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