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 Activity-dependent changes in synaptic protein complex composition are consistent in different detergents despite differential solubility

2019 ◽  
Author(s):  
Jonathan D. Lautz ◽  
Edward P. Gniffke ◽  
Emily A. Brown ◽  
Karen B. Immendorf ◽  
Ryan D. Mendel ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Synaptic Activity ◽  
Synaptic Proteins ◽  
Size Exclusion ◽  
Synaptic Protein ◽  
Exclusion Chromatography ◽  
Associated Proteins ◽  
Dependent Protein ◽  
Activity Dependent

AbstractAt the post-synaptic density (PSD), large protein complexes dynamically form and dissociate in response to synaptic activity, comprising the biophysical basis for learning and memory. The use of detergents to both isolate the PSD fraction and release its membrane-associated proteins complicates studies of these activity-dependent protein interaction networks, because detergents can simultaneously disrupt the very interactions under study. Despite widespread recognition that different detergents yield different experimental results, the effect of detergent on activity-dependent synaptic protein complexes has not been rigorously examined. Here, we characterize the effect of three detergents commonly used to study synaptic proteins on activity-dependent protein interactions. We first demonstrate that SynGAP-containing interactions are more abundant in 1% Deoxycholate (DOC), while Shank-, Homer-and mGluR5-containing interactions are more abundant in 1% NP-40 or Triton. All interactions were detected preferentially in high molecular weight (HMW) complexes generated by size exclusion chromatography, although the detergent-specific abundance of proteins in HMW fractions did not correlate with the abundance of detected interactions. Activity-dependent changes in protein complexes were consistent across detergent types, suggesting that detergents do not isolate distinct protein pools with unique behaviors. However, detection of activity-dependent changes is more or less feasible in different detergents due to baseline solubility. Collectively, our results demonstrate that detergents affect the solubility of individual proteins, but activity-dependent changes in protein interactions, when detectable, are consistent across detergent types.

scholarly journals Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 in Photosystem II

2017 ◽  
Vol 114 (9) ◽  
pp. 2224-2229 ◽  
Author(s):  
Daniel A. Weisz ◽  
Haijun Liu ◽  
Hao Zhang ◽  
Sundarapandian Thangapandian ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Photosystem Ii ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Docking ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Cytochrome B559 ◽  
Reaction Center Complex ◽  
Assembly Intermediate

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b559, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b559, in close proximity to the QB site. Protein–protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the “mass tags” selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein–protein interactions in membrane protein complexes.

scholarly journals Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrinβ1

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Cordula Klockenbusch ◽  
Juergen Kast
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Interaction Analysis ◽  
Protein Complexes ◽  
Human Platelets ◽  
Jurkat Cells ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
High Molecular Weight Complex ◽  
Spectrometry Analysis

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrinβ1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrinβ1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrinβ1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrinβ1,α4 andα6 orβ1,α6,α2, andα5, respectively.

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.

scholarly journals Structure-based validation can drastically under-estimate error rate in proteome-wide cross-linking mass spectrometry studies

2019 ◽  
Author(s):  
Kumar Yugandhar ◽  
Ting-Yi Wang ◽  
Shayne D. Wierbowski ◽  
Elnur Elyar Shayhidin ◽  
Haiyuan Yu
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Error Rates ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
3D Structures ◽  
Cross Links ◽  
Assess Quality ◽  
Almost All

AbstractRecent, rapid advances in cross-linking mass spectrometry (XL-MS) has enabled detection of novel protein-protein interactions and their structural dynamics at the proteome scale. Given the importance and scale of the novel interactions identified in these proteome-wide XL-MS studies, thorough quality assessment is critical. Almost all current XL-MS studies validate cross-links against known 3D structures of representative protein complexes. However, current structure validation approach only includes cross-links where both peptides mapped to the 3D structures. Here we provide theoretical and experimental evidence demonstrating this approach can drastically underestimate error rates for proteome-wide XL-MS datasets. Addressing current shortcomings, we propose and demonstrate a comprehensive set of four metrics, including orthogonal experimental validation to thoroughly assess quality of proteome-wide XL-MS datasets.

scholarly journals RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

2021 ◽  
Vol 7 (1) ◽  
pp. 11 ◽  
Author(s):  
André P. Gerber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Cell Protein ◽  
Transcriptional Regulatory Networks ◽  
Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

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