Simplified Protocol for Cross-linking Mass Spectrometry Using the MS-Cleavable Cross-linker DSBU with Efficient Cross-link Identification

2018 ◽  
Vol 90 (18) ◽  
pp. 10990-10999 ◽  
Author(s):  
Dongqing Pan ◽  
Andreas Brockmeyer ◽  
Franziska Mueller ◽  
Andrea Musacchio ◽  
Tanja Bange
Keyword(s):  
Mass Spectrometry ◽  
Cross Linking ◽  
Cross Link ◽  
Cross Linker

Obtention and Characterization of Collagen and Chitosan Based Cements for Bone Regerneration. Part 1: Extraction and Characterization of Collagen

2014 ◽  
Vol 775-776 ◽  
pp. 29-33 ◽  
Author(s):  
Karine Cappucio de Castro ◽  
Ivan Silva Prado ◽  
Moacyr Clemente Júnior ◽  
Sylma Carvalho Maestrelli ◽  
Neide Aparecida Mariano ◽  
...  
Keyword(s):  
Cross Linking ◽  
Infrared Analysis ◽  
Fiber Structure ◽  
Cross Link ◽  
Scanning Calorimetry ◽  
High Mechanical Strength ◽  
Structure Characteristics ◽  
Cross Linker ◽  
High Degree

Several cements are used as biomaterials. Biopolymers such as chitosan and collagen exhibit excellent biocompatibility and can be used in the remodeling of bone tissue. The cement must have high mechanical strength and compatibility with original tissue. In this context, the objective of this study was to extract, characterize and cross-link collagen from bovine tendon, forlater associate it with chitosan and calcium phosphate to obtain cements for bone regeneration. Glutaraldehyde was used as cross-linker in 0.1, 0.5, 1.0 and 10% concentration. Infrared analysis confirmed the presence of functional groups characteristic of collagen, whereas the capacity of water absorption decreased with the increasing of cross-linking degree. Denaturation temperatures of collagen samples were obtained by Differential Scanning Calorimetry and Scanning Electron Microscopy showed the fiber structure characteristics of collagen, which were more organized for high degree of cross-linking samples.

scholarly journals Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking

2016 ◽  
Vol 88 (20) ◽  
pp. 10215-10222 ◽  
Author(s):  
Jayanta K. Chakrabarty ◽  
Aishwarya G. Naik ◽  
Michael B. Fessler ◽  
Gerhard R. Munske ◽  
Saiful M. Chowdhury
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Cross Linking ◽  
Tandem Mass ◽  
High Confidence ◽  
New Approach ◽  
Cross Linker ◽  
Protein Cross Linking

scholarly journals A Fast Lysine Cross-linker DOPA Enables Mass Spectrometry Analyses of Protein Unfolding and Weak Protein-protein Interactions

2020 ◽  
Author(s):  
Jian-Hua Wang ◽  
Yu-Liang Tang ◽  
Rohit Jain ◽  
Fan Xiao ◽  
Zhou Gong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Guanidine Hydrochloride ◽  
Rnase A ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Protein Structure Analysis ◽  
Cross Linker

AbstractChemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has become a widely used method for protein structure analysis. Central to this technology are chemical cross-linkers. The most popular cross-linkers are N-hydroxysuccinimide (NHS) esters, which react with protein amino groups relatively slowly over 10 minutes or more while in competition with the hydrolysis reaction of NHS esters. To improve the speed of cross-linking, we developed a new class of amine-selective and non-hydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA can cross-link proteins in 10 seconds under near physiological conditions, which is 60 times faster than the NHS ester cross-linker DSS. DOPA also works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Further, DOPA-mediated pulse cross-linking captured the dynamic conformational changes associated with RNase A unfolding. Lastly, DOPA outperformed DSS at capturing weak but specific protein-protein interactions.

scholarly journals MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity

2019 ◽  
Vol 19 (3) ◽  
pp. 554-568 ◽  
Author(s):  
Kumar Yugandhar ◽  
Ting-Yi Wang ◽  
Alden King-Yung Leung ◽  
Michael Charles Lanz ◽  
Ievgen Motorykin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Search Engine ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Cross Linking ◽  
Interaction Patterns ◽  
Protein Protein Interactions ◽  
Cross Link ◽  
Wide Cross ◽  
Cross Links

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a “MS2-centric” approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics [“fraction of mis-identifications” (FMI) and “fraction of interprotein cross-links from known interactions” (FKI)]. We then address this problem, by designing a novel “MS3-centric” approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.

scholarly journals Influence of the chemical structure of cross-linking agents on properties of thermally reversible networks

2016 ◽  
Vol 88 (12) ◽  
pp. 1103-1116 ◽  
Author(s):  
Lorenzo Massimo Polgar ◽  
Robin R.J. Cerpentier ◽  
Gijs H. Vermeij ◽  
Francesco Picchioni ◽  
Martin van Duin
Keyword(s):  
Chemical Structure ◽  
Chemical Conversion ◽  
Rubber Matrix ◽  
Cross Linking ◽  
Cross Link ◽  
Molar Amount ◽  
Link Density ◽  
Cross Link Density ◽  
Cross Linker ◽  
The Cross

Abstract It is well-known that the properties of cross-linked rubbers are strongly affected by the cross-link density. In this work it is shown that for thermoreversibly cross-linked elastomers, the type and length of the cross-linker also have a significant effect. A homologous series of diamine and bismaleimide cross-linkers was used to cross-link maleic-anhydride-grafted EPM irreversibly and furan-modified EPM thermoreversibly, respectively. Bismaleimide cross-linkers with a polarity close to that of EPM and a relatively low melting point have a better solubility in the rubber matrix, which results in higher chemical conversion and, thus, higher cross-link densities at the same molar amount of cross-linker. Samples cross-linked with different spacers (aromatic and aliphatic spacers of different lengths) were compared at the same cross-link density to interpret the effects on the material properties. The rigid character of the short aliphatic and the aromatic cross-linkers accounts for the observed increase in hardness, Young´s modulus and tensile strength with respect to the longer, more flexible aliphatic cross-linkers. In conclusion, the structure of the cross-linking agent can be considered as an alternative variable in tuning the rubber properties, especially for thermoreversibly cross-linked rubber.

scholarly journals The microtubule cross-linker Feo controls the midzone stability, motor composition, and elongation of the anaphase B spindle in Drosophila embryos

2015 ◽  
Vol 26 (8) ◽  
pp. 1452-1462 ◽  
Author(s):  
Haifeng Wang ◽  
Ingrid Brust-Mascher ◽  
Jonathan M. Scholey
Keyword(s):  
Chromosome Segregation ◽  
Cross Linking ◽  
Cross Link ◽  
Spindle Poles ◽  
Cross Linker ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Drosophila Embryos ◽  
Poleward Flux

Chromosome segregation during anaphase depends on chromosome-to-pole motility and pole-to-pole separation. We propose that in Drosophila embryos, the latter process (anaphase B) depends on a persistent kinesin-5–generated interpolar (ip) microtubule (MT) sliding filament mechanism that “engages” to push apart the spindle poles when poleward flux is turned off. Here we investigated the contribution of the midzonal, antiparallel MT-cross-linking nonmotor MAP, Feo, to this “slide-and-flux-or-elongate” mechanism. Whereas Feo homologues in other systems enhance the midzone localization of the MT-MT cross-linking motors kinesin-4, -5 and -6, the midzone localization of these motors is respectively enhanced, reduced, and unaffected by Feo. Strikingly, kinesin-5 localizes all along ipMTs of the anaphase B spindle in the presence of Feo, including at the midzone, but the antibody-induced dissociation of Feo increases kinesin-5 association with the midzone, which becomes abnormally narrow, leading to impaired anaphase B and incomplete chromosome segregation. Thus, although Feo and kinesin-5 both preferentially cross-link MTs into antiparallel polarity patterns, kinesin-5 cannot substitute for loss of Feo function. We propose that Feo controls the organization, stability, and motor composition of antiparallel ipMTs at the midzone, thereby facilitating the kinesin-5–driven sliding filament mechanism underlying proper anaphase B spindle elongation and chromosome segregation.

scholarly journals Why Are Two Different Cross-linkers Necessary for Actin Bundle Formation In Vivo and What Does Each Cross-link Contribute?

1998 ◽  
Vol 143 (1) ◽  
pp. 121-133 ◽  
Author(s):  
Lewis G. Tilney ◽  
Patricia S. Connelly ◽  
Kelly A. Vranich ◽  
Michael K. Shaw ◽  
Gregory M. Guild
Keyword(s):  
Potassium Iodide ◽  
Actin Filaments ◽  
Cross Linking ◽  
Wild Type ◽  
Cross Link ◽  
Actin Bundle ◽  
Cross Linker ◽  
Three Phases

In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus ∼700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur.

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 Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry

2018 ◽  
Vol 115 (44) ◽  
pp. 11162-11167 ◽  
Author(s):  
Bing Yang ◽  
Haifan Wu ◽  
Paul D. Schnier ◽  
Yansheng Liu ◽  
Jun Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Current Method ◽  
Thermal Fluctuations ◽  
Limited Range ◽  
Live Cells ◽  
Cross Linking ◽  
Cross Links ◽  
Cross Linker ◽  
Sulfonyl Fluoride ◽  
Chemical Cross Linking

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a “plant-and-cast” cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues “planting” the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then “cast” across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme–substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

The Isotope-Labeled, MS-Cleavable Cross-Linker Disuccinimidyl Dibutyric Urea for Improved Cross-Linking/Mass Spectrometry Studies

2019 ◽  
Vol 31 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Christian H. Ihling ◽  
Patrizia Springorum ◽  
Claudio Iacobucci ◽  
Christoph Hage ◽  
Michael Götze ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cross Linking ◽  
Cross Linker
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