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 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.

EXPERIMENTAL VALIDATION OF THE CHARLESBY AND HORIKX MODELS APPLIED TO DE-VULCANIZATION OF SULFUR AND PEROXIDE VULCANIZATES OF NR AND EPDM

2016 ◽  
Vol 89 (4) ◽  
pp. 671-688 ◽  
Author(s):  
M. A. L. Verbruggen ◽  
L. van der Does ◽  
W. K. Dierkes ◽  
J. W. M. Noordermeer
Keyword(s):  
Experimental Validation ◽  
Main Chain ◽  
Sol Gel ◽  
Chain Scission ◽  
Cross Linking ◽  
Cross Link ◽  
Practical Reality ◽  
Cross Links ◽  
The Cross ◽  
Theoretical Considerations

ABSTRACT The theoretical model developed by Charlesby to quantify the balance between cross-links creation of polymers and chain scission during radiation cross-linking and further modifications by Horikx to describe network breakdown from aging were merged to characterize the balance of both types of scission on the development of the sol content during de-vulcanization of rubber networks. There are, however, disturbing factors in these theoretical considerations vis-à-vis practical reality. Sulfur- and peroxide-cured NR and EPDM vulcanizates were de-vulcanized under conditions of selective cross-link and random main-chain scissions. Cross-link scission was obtained using thiol-amine reagents for selective cleavage of sulfur cross-links. Random main-chain scission was achieved by heating peroxide vulcanizates of NR with diphenyldisulfide, a method commonly employed for NR reclaiming. An important factor in the analyses of these experiments is the cross-linking index. Its value must be calculated using the sol fraction of the cross-linked network before de-vulcanization to obtain reliable results. The values for the cross-linking index calculated with sol-gel data before de-vulcanization appear to fit the experimentally determined modes of network scission during de-vulcanization very well. This study confirms that the treatment of de-vulcanization data with the merged Charlesby and Horikx models can be used satisfactorily to characterize the de-vulcanization of NR and EPDM vulcanizates.

scholarly journals Bound (“Glassy”) Rubber as a Free Radical Cross-linked Rubber Layer on a Carbon Black

Materials ◽  
2018 ◽  
Vol 11 (10) ◽  
pp. 1992 ◽  
Author(s):  
Alexey Kondyurin ◽  
Anastasia Eliseeva ◽  
Alexander Svistkov
Keyword(s):  
Free Radical ◽  
Carbon Black ◽  
Ion Beam ◽  
Cross Linking ◽  
Carbon Filler ◽  
Free Radical Reactions ◽  
Rubber Layer ◽  
Ion Beam Implantation ◽  
Cross Links ◽  
The Cross

A model of rubber with a cross-linked rubber layer on a carbon black filler has been proposed. The cross-links are the result of free radical reactions generated by carbon atoms with unpaired electrons at the edge of graphitic sheets in a carbon black filler. The experimental study of the cross-linking reactions in polyisoprene was done on a flat carbonized surface after ion beam implantation. The cross-linking process in the polyisoprene macromolecules between two particles was simulated. The model with a cross-linked rubber layer on a carbon filler as a “glassy layer” explains the mechanical properties of the rubber materials.

Sorption of Caustic Solution by Formaldehyde-Crosslinked Cottons

1969 ◽  
Vol 39 (11) ◽  
pp. 1023-1030 ◽  
Author(s):  
Edith Honold ◽  
Stanley P. Rowland ◽  
James N. Grant
Keyword(s):  
Caustic Solution ◽  
Cotton Fibers ◽  
Cross Linking ◽  
Centrifuge Test ◽  
Fiber Structure ◽  
Formaldehyde Content ◽  
Related Data ◽  
Wide Range ◽  
Cross Links ◽  
The Cross

Differences in the ability of formaldehyde-crosslinked cotton fibers to swell are demonstrated in terms of alkali centrifuge values (ACV), i.e., the sorption of caustic solution of mercerizing strength. The wide range in ACV (310–50) emphasizes the extremes in sorptivity that can be achieved by differences in formaldehyde content and in method of introducing the cross links. In general, the ACV decreases with increasing formaldehyde content. However, ACV higher than that of the noncross-linked control cotton are reached for those samples in which a low percentage of formaldehyde was introduced into water-swollen fibers. Various hypotheses, based on ACV and related data, are presented pertaining to the alterations in fiber structure during the cross-linking processes and during the alkali swelling centrifuge test

scholarly journals p-NN′-phenylenebismaleimide, a specific cross-linking agent for F-actin

1978 ◽  
Vol 175 (3) ◽  
pp. 1023-1032 ◽  
Author(s):  
P Knight ◽  
G Offer
Keyword(s):  
Quantitative Analysis ◽  
Sodium Dodecyl Sulphate ◽  
Actin Filaments ◽  
Sodium Dodecyl ◽  
Cysteine Residue ◽  
Cross Linking ◽  
Maximum Value ◽  
Cross Links ◽  
The Cross ◽  
Hydrolysis Of

Covalent cross-links can be inserted between the subunits of F-actin by using p-NN′-phenylenebismaleimide. Cross-linking reaches its maximum value when one molecule of reagent has reacted with each actin subunit. p-NN′-Phenylenebismaleimide reacts initially with a cysteine residue on one subunit, the slower cross-linking reaction involving a lysine residue on a neighbouring subunit. Hydrolysis of the actin-bound reagent limits the extent of cross-linking. Quantitative analysis of the amounts of cross-linked oligomers seen on polyacrylamide gels containing sodium dodecyl sulphate suggests that neither the binding of the reagent to actin nor the formation of cross-links introduces strain into the structure. The cross-links do not join together different F-actin filaments, and evidence is presented that suggests that the cross-links join subunits of the same long-pitched helix.

scholarly journals Chemistry of collagen cross-linking: biochemical changes in collagen during the partial mineralization of turkey leg tendon

1997 ◽  
Vol 322 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Lynda KNOTT ◽  
John F. TARLTON ◽  
Allen J. BAILEY
Keyword(s):  
Collagen Matrix ◽  
Cross Linking ◽  
Cross Link ◽  
Lysine Residues ◽  
Collagen Mineralization ◽  
Cross Links ◽  
The Cross ◽  
Calcified Tissues ◽  
Turkey Leg Tendon ◽  
Collagen Cross Linking

With age, the proximal sections of turkey leg tendons become calcified, and this phenomenon has led to their use as a model for collagen mineralization. Mineralizing turkey leg tendon was used in this study to characterize further the composition and cross-linking of collagen in calcified tissues. The cross-link profiles of mineralizing collagen are significantly different from those of other collagenous matrices with characteristically low amounts of hydroxylysyl-pyridinoline and the presence of lysyl-pyridinoline and pyrrolic cross-links. However, the presence of the immature cross-link precursors previously reported in calcifying tissues was not supported in the present study, and was found to be due to the decalcification procedure using EDTA. Analysis of tendons from young birds demonstrated differences in the cross-link profile which indicated a higher level of hydroxylation of specific triple-helical lysines involved in cross-linking of the proximal tendon. This may be related to later calcification, suggesting that this part of the tendon is predestined to be calcified. The minimal changes in lysyl hydroxylation in both regions of the tendon with age were in contrast with the large changes in the cross-link profile, indicating differential hydroxylation of the helical and telopeptide lysine residues. Changes with age in the collagen matrix, its turnover and thermal properties in both the proximal and distal sections of the tendon clearly demonstrate that a new and modified matrix is formed throughout the tendon, and that a different type of matrix is formed at each site.

scholarly journals Ultrasound-Assisted Transglutaminase Catalysis of the Cross-Linking and Microstructure of αs-Casein, β-Casein and κ-Casein

Processes ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1630
Author(s):  
Chun-Chi Chen ◽  
Liang-Yu Chen ◽  
Wen-Tai Li ◽  
Ken-Lin Chang ◽  
Hsien-Wei Tseng ◽  
...  
Keyword(s):  
Particle Size Analysis ◽  
Size Analysis ◽  
Cross Linking ◽  
Comprehensive Utilization ◽  
Transmission Electron ◽  
Microscopy Investigation ◽  
Electron Microscopy Investigation ◽  
Sds Page ◽  
The Cross ◽  
The Individual

The effects of ultrasonic treatment (UT)-assisted transglutaminase (TGase) catalysis on the physicochemical properties of individual αs-casein (αs-CN), β-casein (β-CN), and κ-casein (κ-CN) were investigated. After 60 min of incubation at 30 °C, αs-CN, β-CN, and κ-CN were cross-linked with TGase (6.0 units/mL), and high molecular weight polymers (>200 kDa) were formed. The use of TGase in conjunction with UT (20 kHz, power of 400 W, and amplitude 20%) led to an increase in the rate of αs-CN, β-CN, and κ-CN polymerization compared to the individual casein that contained TGase but did not undergo UT. SDS-PAGE scrutiny showed that the intensities of αs-CN, β-CN, and κ-CN incubation with regard to TGase and UT at 30 °C for 60 min noticeably decreased to 5.66 ± 0.39, 3.97 ± 0.43, and 26.07 ± 1.18%, respectively (p < 0.05). Particle size analysis results indicated that the molecule size appropriation for the cross-linking of αs-CN, β-CN, and κ-CN ranged from 6000 to 10,000 nm after 60 min incubation with TGase and UT. Transmission electron microscopy investigation showed network structures of cross-linking αs-CN, β-CN, and κ-CN were formed from αs-CN, β-CN, and κ-CN, respectively. As our results show, the comprehensive utilization of TGase and UT will be a superior method for the polymerization of αs-CN, β-CN, and κ-CN.

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 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.

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