scholarly journals Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

10.3791/57902 ◽  
2018 ◽  
Author(s):  
Alexandra J. Machen ◽  
Pierce T. O'Neil ◽  
Bradley L. Pentelute ◽  
Maria T. Villar ◽  
Antonio Artigues ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Protein Complexes ◽  
Biolayer Interferometry

Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes

2021 ◽  
pp. 217-232
Author(s):  
Lucía Quintana-Gallardo ◽  
Moisés Maestro-López ◽  
Jaime Martín-Benito ◽  
Miguel Marcilla ◽  
Daniel Rutz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Structural Characterization ◽  
Protein Complexes ◽  
Cross Linking

scholarly journals Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa

2012 ◽  
Vol 11 (11) ◽  
pp. 1430-1441 ◽  
Author(s):  
Esther van Duijn ◽  
Ioana M. Barbu ◽  
Arjan Barendregt ◽  
Matthijs M. Jore ◽  
Blake Wiedenheft ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Ion Mobility ◽  
Protein Complexes ◽  
Acquired Resistance ◽  
Native Mass Spectrometry ◽  
Ion Mobility Mass Spectrometry ◽  
E Coli ◽  
Ribonucleoprotein Complexes ◽  
Specific Association

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

scholarly journals Classification of Single Particles from Human Cell Extract Reveals Distinct Structures

2018 ◽  
Author(s):  
Eric J. Verbeke ◽  
Anna L. Mallam ◽  
Kevin Drew ◽  
Edward M. Marcotte ◽  
David W. Taylor
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Protein Identification ◽  
Size Structure ◽  
Protein Complexes ◽  
Cell Extract ◽  
Particle Analysis ◽  
Proteomics Data ◽  
Genomics And Proteomics

SummaryMulti-protein complexes are necessary for nearly all cellular processes, and understanding their structure is required for elucidating their function. Current high-resolution strategies in structural biology are effective, but lag behind other fields (e.g. genomics and proteomics) due to their reliance on purified samples rather than characterizing heterogeneous mixtures. Here, we present a method combining single particle analysis by electron microscopy with protein identification by mass spectrometry to structurally characterize macromolecular complexes from extracts of human cells. We obtain three-dimensional structures of native proteasomes directly from ab initio classification of a heterogeneous mixture of protein complexes. In addition, we find an ~1 MDa size structure of unknown composition and reference our proteomics data to suggest possible identities. Our study shows the power of using a shotgun approach to electron microscopy (shotgun EM) when coupled with mass spectrometry as a tool to uncover the structures of macromolecular machines in parallel.

Electron Microscopy and image analysis of nucleo-protein complexes

1994 ◽  
Vol 52 ◽  
pp. 88-89
Author(s):  
E. H. Egelman ◽  
X. Yu
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Normal Cell ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Chromosomal Rearrangements ◽  
Reca Protein ◽  
E Coli ◽  
Helical Polymer ◽  
Specific Protease

The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. The unusual filament formed by the RecA protein on DNA has not previously been shown to exist outside of bacteria. Within this filament, the 36 Å pitch of B-form DNA is extended to about 95 Å, the pitch of the RecA helix. We have now establishedthat similar nucleo-protein complexes are formed by bacteriophage and yeast proteins, and availableevidence suggests that this structure is universal across all of biology, including humans. Thus, understanding the function of the RecA protein will reveal basic mechanisms, in existence inall organisms, that are at the foundation of general genetic recombination and repair.Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer.

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Mass Spectrometry for Proteomics and Recent Developments in ESI, MALDI and other Ionization Methodologies

2019 ◽  
Vol 16 (4) ◽  
pp. 267-276
Author(s):  
Qurat ul Ain Farooq ◽  
Noor ul Haq ◽  
Abdul Aziz ◽  
Sara Aimen ◽  
Muhammad Inam ul Haq
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Ionization ◽  
New Technologies ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Desorption Electrospray Ionization ◽  
High Throughput Analysis ◽  
Enhanced Sensitivity ◽  
Recent Developments ◽  
The Way

Background: Mass spectrometry is a tool used in analytical chemistry to identify components in a chemical compound and it is of tremendous importance in the field of biology for high throughput analysis of biomolecules, among which protein is of great interest. Objective: Advancement in proteomics based on mass spectrometry has led the way to quantify multiple protein complexes, and proteins interactions with DNA/RNA or other chemical compounds which is a breakthrough in the field of bioinformatics. Methods: Many new technologies have been introduced in electrospray ionization (ESI) and Matrixassisted Laser Desorption/Ionization (MALDI) techniques which have enhanced sensitivity, resolution and many other key features for the characterization of proteins. Results: The advent of ambient mass spectrometry and its different versions like Desorption Electrospray Ionization (DESI), DART and ELDI has brought a huge revolution in proteomics research. Different imaging techniques are also introduced in MS to map proteins and other significant biomolecules. These drastic developments have paved the way to analyze large proteins of >200kDa easily. Conclusion: Here, we discuss the recent advancement in mass spectrometry, which is of great importance and it could lead us to further deep analysis of the molecules from different perspectives and further advancement in these techniques will enable us to find better ways for prediction of molecules and their behavioral properties.

scholarly journals Application of Cryo-Electron Microscopy on Drug Discovery

2021 ◽  
Vol 27 (S1) ◽  
pp. 3250-3250
Author(s):  
Viswanath Vittaladevaram ◽  
Kranthi Kuruti
Keyword(s):  
Electron Microscopy ◽  
Protein Complexes ◽  
Lead Discovery ◽  
Biologically Relevant ◽  
Biological Targets ◽  
3 Dimensional ◽  
Drug Molecules ◽  
Cryo Electron Microscopy ◽  
Therapeutic Molecules ◽  
Novel Drug

AbstractThe key aspect for development of novel drug molecules is to perform structural determination of target molecule associated with its ligand. One such tool that provides insights towards structure of molecule is Cryo-electron microscopy which covers biological targets that are intractable. Examination of proteins can be carried out in native state, as the samples are frozen at -175 degree Celsius i.e. cryogenic temperatures. In addition to this, there were no limits for molecular and functional structures of proteins that can be imagined in 3-dimensional form. This includes ligands which unravel mechanisms that are biologically relevant. This will enable to better understand the mechanisms that are used for development of new therapeutics. Application of Cryo-electron microscopy is not limited to protein complexes and is considered as non-specific. Intervention of Cryo-EM would allow to analyse the structures and also able to dissect the interaction with therapeutic molecules. The study determines the usage of cryo-EM for providing resolutions that are acceptable for lead discovery. It also provides support for lead optimization and also for discovery of vaccines and therapeutics.

Sign in / Sign up

Export Citation Format

Share Document