Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells

Methods ◽  
2015 ◽  
Vol 89 ◽  
pp. 121-127 ◽  
Author(s):  
Christine Piotrowski ◽  
Christian H. Ihling ◽  
Andrea Sinz
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Amino Acids ◽  
Cross Linking ◽  
Escherichia Coli Cells ◽  
Model Protein ◽  
The Cross

Overproduction of noncanonical amino acids by Escherichia coli cells

Microbiology ◽  
2007 ◽  
Vol 76 (6) ◽  
pp. 712-718 ◽  
Author(s):  
E. V. Sycheva ◽  
T. A. Yampol’skaya ◽  
E. S. Preobrajenskaya ◽  
A. E. Novikova ◽  
N. G. Matrosov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Noncanonical Amino Acids ◽  
Escherichia Coli Cells

scholarly journals Bacterial protein translocation requires only one copy of the SecY complex in vivo

2012 ◽  
Vol 198 (5) ◽  
pp. 881-893 ◽  
Author(s):  
Eunyong Park ◽  
Tom A. Rapoport
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Protein Translocation ◽  
Cross Linking ◽  
Bacterial Protein ◽  
Oligomeric State ◽  
E Coli ◽  
Cotranslational Translocation ◽  
Escherichia Coli Cells

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation.

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 Stitching the synapse: Cross-linking mass spectrometry into resolving synaptic protein interactions

2020 ◽  
Vol 6 (8) ◽  
pp. eaax5783 ◽  
Author(s):  
M. A. Gonzalez-Lozano ◽  
F. Koopmans ◽  
P. F. Sullivan ◽  
J. Protze ◽  
G. Krause ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Conformational Dynamics ◽  
Synaptic Proteins ◽  
Cross Linking ◽  
Synaptic Protein ◽  
Protein Partners ◽  
Associated Proteins ◽  
Model Protein

Synaptic transmission is the predominant form of communication in the brain. It requires functionally specialized molecular machineries constituted by thousands of interacting synaptic proteins. Here, we made use of recent advances in cross-linking mass spectrometry (XL-MS) in combination with biochemical and computational approaches to reveal the architecture and assembly of synaptic protein complexes from mouse brain hippocampus and cerebellum. We obtained 11,999 unique lysine-lysine cross-links, comprising connections within and between 2362 proteins. This extensive collection was the basis to identify novel protein partners, to model protein conformational dynamics, and to delineate within and between protein interactions of main synaptic constituents, such as Camk2, the AMPA-type glutamate receptor, and associated proteins. Using XL-MS, we generated a protein interaction resource that we made easily accessible via a web-based platform (http://xlink.cncr.nl) to provide new entries into exploration of all protein interactions identified.

scholarly journals Modulation of Entrapment Efficiency and In Vitro Release Properties of BSA-Loaded Chitosan Microparticles Cross-Linked with Citric Acid as a Potential Protein–Drug Delivery System

Materials ◽  
2020 ◽  
Vol 13 (8) ◽  
pp. 1989 ◽  
Author(s):  
Natalia Sedyakina ◽  
Andrey Kuskov ◽  
Kelly Velonia ◽  
Nataliya Feldman ◽  
Sergey Lutsenko ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Citric Acid ◽  
In Vitro Release ◽  
Entrapment Efficiency ◽  
Cross Linking ◽  
Release Mechanism ◽  
Model Protein ◽  
Cross Linker ◽  
The Cross

Microparticles, aimed for oral protein and peptide drug delivery, were prepared via emulsion cross-linking using citric acid as cross-linker and polyglycerol polyricinoleate as surfactant. A comparative study of the interaction between chitosan and citric acid and its effect on the resulting microparticle properties was performed using different chitosan-to-cross-linker mass ratios and pH-values during fabrication of the microparticles. Non-cross-linked and cross-linked microparticles were studied in terms of size (4–12 μm), zeta potential (−15.7 to 12.8 mV), erosion (39.7–75.6%), a model protein encapsulation efficiency (bovine serum albumin) (6.8–27.6%), and loading capacity (10.4–40%). Fourier transform infrared spectroscopy and X-ray diffraction confirmed the ionic interaction between the protonated amine groups of chitosan and the carboxylate ions of the cross-linking agent. Scanning electron microscopy revealed that the non-cross-linked microparticles had an uneven shape with wrinkled surfaces, while the cross-linked formulations were spherical in shape with smooth surfaces. On the basis of these data, the role of the surfactant and microparticle structure on the release mechanism was proposed. Control of the microparticle shape and release mechanisms is expected to be crucial in developing carriers for the controlled delivery of proteins and peptides.

scholarly journals Synthesis of two new enrichable and MS-cleavable cross-linkers to define protein–protein interactions by mass spectrometry

2015 ◽  
Vol 13 (17) ◽  
pp. 5030-5037 ◽  
Author(s):  
Anthony M. Burke ◽  
Wynne Kandur ◽  
Eric J. Novitsky ◽  
Robyn M. Kaake ◽  
Clinton Yu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
The Cross

The cross-linking Mass Spectrometry (XL-MS) technique extracts structural information from protein complexes without requiring highly purified samples, crystallinity, or large amounts of material.

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