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

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.

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

10.1101/617654 ◽

2019 ◽

Cited By ~ 5

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1620360114 ◽

2017 ◽

Vol 114 (9) ◽

pp. 2224-2229 ◽

Cited By ~ 18

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.

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Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

10.1101/450981 ◽

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.

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MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity

Molecular & Cellular Proteomics ◽

10.1074/mcp.tir119.001847 ◽

2019 ◽

Vol 19 (3) ◽

pp. 554-568 ◽

Cited By ~ 8

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.

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Genetically encoded selective cross-linkers and emerging applications

Biochemical Society Transactions ◽

10.1042/bst20200508 ◽

2020 ◽

Vol 48 (4) ◽

pp. 1807-1817

Author(s):

Haiyan Ren

Keyword(s):

Protein Interactions ◽

Unnatural Amino Acids ◽

Protein Complexes ◽

Living Cells ◽

Cross Linking ◽

Protein Protein Interactions ◽

New Class ◽

Selective Reaction ◽

Biological Studies ◽

Lower Background

There has been a large amount of interest in the development of genetically encoded cross-linkers that target functional groups naturally present in cells. Recently, a new class of unnatural amino acids that specifically react with target residues were developed and genetically incorporated. The selective reaction shows higher cross-linking efficiency, lower background and predictable cross-linking sites. It has been applied to enhance protein/peptide stability, pinpoint protein–protein interactions, stabilize protein complexes, engineer covalent protein inhibitors, identify phosphatases in living cells, etc. These new covalent linkages provide excellent new tools for protein engineering and biological studies. Their applications in biotherapy will provide considerable opportunities for innovating and improving biomolecular medicines.

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

Organic & Biomolecular Chemistry ◽

10.1039/c5ob00488h ◽

2015 ◽

Vol 13 (17) ◽

pp. 5030-5037 ◽

Cited By ~ 20

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

10.17504/protocols.io.b2f6qbre ◽

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.

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

10.1101/526897 ◽

2019 ◽

Cited By ~ 1

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.

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

10.1101/094961 ◽

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.

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Development of a compact alkynyl-enrichable crosslinker for in-depth in-vivo crosslinking analysis

10.1101/2021.07.30.454285 ◽

2021 ◽

Author(s):

Hang Gao ◽

Li Li Zhao ◽

Qun Zhao ◽

Hua Li Zhang ◽

Feng Bao Zhao ◽

...

Keyword(s):

Protein Interactions ◽

Large Scale ◽

High Efficiency ◽

Protein Complexes ◽

Protein Structures ◽

High Sensitivity ◽

Protein Protein Interactions ◽

Cross Links ◽

First Time

Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to capture the dynamic information of protein complexes with high sensitivity, throughput and sample universality. To advance the study of in-vivo protein structures and protein-protein interactions on the large scale, a new alkynyl-enrichable crosslinker was developed with high efficiency of membrane penetration, reactivity and enrichment. The crosslinker was successfully used for in-vivo crosslinking of intact human cells, resulting in 6820 non-redundant crosslinks identified at a false discovery rate (FDR) of 1% using pLink 2.0, which 4898 (71.8%) of the cross-links were assigned as intraprotein and 1922 (28.2%) were interprotein links. To our knowledge, this is also the first time to realize the in-vivo crosslinking with a non-cleavable cross-linker for homo species cells.

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