scholarly journals In culture cross-linking of bacterial cells reveals proteome scale dynamic protein-protein interactions at the peptide level

2016 ◽  
Author(s):  
Luitzen de Jong ◽  
Edward A. de Koning ◽  
Winfried Roseboom ◽  
Hansuk Buncherd ◽  
Martin J. Wanner ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Protein Interactions ◽  
Cross Linking ◽  
Bacterial Cells ◽  
Protein Protein Interactions ◽  
Genome Wide ◽  
Peptide Level ◽  
Complex Protein ◽  
Genome Wide Data ◽  
Cross Links

AbstractIdentification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram positive model bacteriumBacillus subtilisas an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species,Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and β′ subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.

scholarly journals In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein–Protein Interactions at the Peptide Level

2017 ◽  
Vol 16 (7) ◽  
pp. 2457-2471 ◽  
Author(s):  
Luitzen de Jong ◽  
Edward A. de Koning ◽  
Winfried Roseboom ◽  
Hansuk Buncherd ◽  
Martin J. Wanner ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Cross Linking ◽  
Bacterial Cells ◽  
Protein Protein Interactions ◽  
Peptide Level

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 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 Cross-linking of the Endolysosomal System Reveals Flotillin Structures and Putative Cargo

2022 ◽  
Author(s):  
Jasjot Singh ◽  
Hadeer Elhabashy ◽  
Pathma Muthukottiappan ◽  
Markus Stepath ◽  
Martin Eisenacher ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Decisive Role ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Lysosome Biogenesis ◽  
Early Endosomes ◽  
Cross Links ◽  
Related Proteins ◽  
G Protein Coupled

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes, but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyzed lysosomes and early endosomes. Based on the identification of 5,376 cross-links, we investigated protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and heterodimeric/-multimeric structures of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identified >300 proteins presenting putative cargo, and confirm the latrophilin family of adhesion G protein-coupled receptors as substrates for flotillin-dependent endocytosis.

Cross-linking/MS analysis of TBEV and neuroblastoma cells v1

2021 ◽  
Author(s):  
Sarah Barrass ◽  
Lauri I. A. Pulkkinen ◽  
Olli Vapalahti ◽  
Suvi H. Kuivanen ◽  
Maria Anastasina ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Internal Control ◽  
Limited Proteolysis ◽  
Neuroblastoma Cells ◽  
Host Protein ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Cross Links ◽  
Using Data

Virus-host protein-protein interactions are central to viral infection, but are challenging to identify and characterise, especially in complex systems involving intact viruses and cells. Here, we describe a proteome-wide approach to identify virus-host interactions using chemical cross-linking coupled with mass spectrometry. We adsorbed tick-borne encephalitis virus onto metabolically-stalled neuroblastoma cells, covalently cross-linked interacting virus-host proteins, and performed limited proteolysis to release primarily the surface-exposed proteins for analysis by mass spectrometry. Proteins in the sample were identified using data-dependent acquisition mass spectrometry and cross-linked peptides were identified using the software pLink2. Cross-links are validated using the intraviral cross-links as an internal control.

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 MaXLinker: proteome-wide cross-link identifications with high specificity and sensitivity

2019 ◽  
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

ABSTRACTProtein-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 has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. However, the current cross-link search algorithms follow an “MS2-centric” approach and, as a result, suffer from a high rate of mis-identified cross-links (~15%). We address this urgent problem, by designing a novel “MS3-centric” approach for cross-link identification and implemented it as a search engine called MaXLinker. MaXLinker significantly outperforms the current state of the art search engine with up to 18-fold lower false positive rate. Additionally, MaXLinker results in up to 31% more cross-links, demonstrating its superior sensitivity and specificity. Moreover, we performed proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we unveiled the most comprehensive set of 9,319 unique cross-links at 1% false discovery rate, comprising 8,051 intraprotein and 1,268 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 Roles of the 14-3-3 gene family in cotton flowering

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Sang ◽  
Hui Liu ◽  
Bin Ma ◽  
Xianzhong Huang ◽  
Lu Zhuo ◽  
...  
Keyword(s):  
Gene Family ◽  
Protein Interactions ◽  
Leucine Zipper ◽  
Expression Patterns ◽  
Bimolecular Fluorescence Complementation ◽  
Structural Motif ◽  
Virus Induced Gene Silencing ◽  
Protein Protein Interactions ◽  
Basic Leucine Zipper ◽  
Genome Wide

Abstract Background In plants, 14-3-3 proteins, also called GENERAL REGULATORY FACTORs (GRFs), encoded by a large multigene family, are involved in protein–protein interactions and play crucial roles in various physiological processes. No genome-wide analysis of the GRF gene family has been performed in cotton, and their functions in flowering are largely unknown. Results In this study, 17, 17, 31, and 17 GRF genes were identified in Gossypium herbaceum, G. arboreum, G. hirsutum, and G. raimondii, respectively, by genome-wide analyses and were designated as GheGRFs, GaGRFs, GhGRFs, and GrGRFs, respectively. A phylogenetic analysis revealed that these proteins were divided into ε and non-ε groups. Gene structural, motif composition, synteny, and duplicated gene analyses of the identified GRF genes provided insights into the evolution of this family in cotton. GhGRF genes exhibited diverse expression patterns in different tissues. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhGRFs interacted with the cotton FLOWERING LOCUS T homologue GhFT in the cytoplasm and nucleus, while they interacted with the basic leucine zipper transcription factor GhFD only in the nucleus. Virus-induced gene silencing in G. hirsutum and transgenic studies in Arabidopsis demonstrated that GhGRF3/6/9/15 repressed flowering and that GhGRF14 promoted flowering. Conclusions Here, 82 GRF genes were identified in cotton, and their gene and protein features, classification, evolution, and expression patterns were comprehensively and systematically investigated. The GhGRF3/6/9/15 interacted with GhFT and GhFD to form florigen activation complexs that inhibited flowering. However, GhGRF14 interacted with GhFT and GhFD to form florigen activation complex that promoted flowering. The results provide a foundation for further studies on the regulatory mechanisms of flowering.

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