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Enhanced protein-protein interaction network construction promoted by in vivo cross-linking

ABSTRACT: Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptides enrichment. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linker, we explored the click chemistry-based enrichment approaches on protein and peptide level with three cleavable click chemistry ligands, respectively. By comparison, the approach of protein-based click chemistry conjugation with acid-cleavable tag was demonstrated to permit the most cross-linked peptides identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518 protein-protein interactions network among 1,871 proteins with wide abundance distribution in cell. Therefore, all these results demonstrated a guideline value of our work for efficient cross-linked peptides enrichment, thus facilitated the in-depth profiling of protein interactome for functional analysis.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Lihua Zhang

PROVIDER: PXD035355 | JPOST Repository | Fri Jul 15 00:00:00 BST 2022

REPOSITORIES: jPOST

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			Action	DRS
	293T-BSP-Pep-click-UV-break-3ul-2_20201214092120.raw	Raw
	293T-BSP-Pep-click-UV-break-3ul-3_20201214121948.raw	Raw
	293T-BSP-Pep-click-UV-break-3ul.raw	Raw
	293T-BSP-Pep-click-acid-break-3ul-2.raw	Raw
	293T-BSP-Pep-click-acid-break-3ul-3.raw	Raw

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Enhanced protein-protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click chemistry enrichment

Project description:Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptides enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click chemistry ligands (acid, reduction, photo) for cross-linked peptides enrichment and release is lacking. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linker, we explored the click chemistry-based enrichment approaches on protein and peptide level with three cleavable click chemistry ligands, respectively. By comparison, the approach of protein-based click chemistry conjugation with acid-cleavable tag was demonstrated to permit the most cross-linked peptides identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518 protein-protein interactions network among 1,871 proteins with wide abundance distribution in cell. Therefore, all these results demonstrated a guideline value of our work for efficient cross-linked peptides enrichment, thus facilitated the in-depth profiling of protein interactome for functional analysis.

2022-07-14 | PXD035317 | JPOST Repository

Development of an sulfoxide-based, CuAAC-enrichable cross-linker

Project description:Cross-linking of BSA with a novel cross-linker. Modification of the cross-linker containing peptides with CuAAC-chemistry to attach a cleavable biotin-derivative. Enrichment with streptavidin-beads.

2019-10-14 | PXD015080 | Pride

Tyrosine/Lysine-Targeting Urazole Cross-Linkers for Protein Structural Analysis

Project description:Chemical cross-linking mass spectrometry (XL-MS) is essential for studying protein structures and interactions. While lysine-targeted cross-linkers dominate current applications, this study introduces two urazole-derived MS-cleavable cross-linkers for tyrosine and lysine residues. A homobifunctional cross-linker (SBT) targets tyrosine via electrochemical click chemistry, while a heterobifunctional cross-linker (SCT) binds both lysine and tyrosine. Their efficiency was validated through reactions with peptides, proteins, and complexes. Additionally, AlphaFold 3-predicted BSA dimer structures were compared with crystallographic data, revealing improved structural coverage. These cross-linkers expand the XL-MS toolkit, enhancing protein structural analysis accuracy and scope.

2025-02-07 | PXD060590 |

Developing In Vivo Cross-linking Mass Spectrometry (XL-MS) to Decipher Protein Interaction Landscapes at the Proteome Scale

Project description:We have developed a new and robust in vivo cross-linking mass spectrometry (XL-MS) that utilizes a multifunctional MS-cleavable cross-linker to enable the efficient capture, enrichment, and identification of in vivo cross-linked peptides at the whole proteome scale.

2021-07-30 | PXD012788 | Pride

HaloTag-based conjugation of proteins to barcoding-oligonucleotides

Project description:We describe the development of a high-sensitivity protein quantification system called HaloTag protein barcoding assay. The assay involves target protein linking to a unique molecule-counting oligonucleotide by click chemistry.

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Universal Enrichment and Sequencing of DNA Modifications Using a Click-Coupled IgG-Protein G System

Project description:Efficient nucleic acid enrichment is pivotal for deciphering epigenetic modifications and disease biomarkers, yet current methods are constrained by insufficient specificity, poor versatility, and high costs. We developed a universal strategy named ‘Click-IP-Seq’ by leveraging the high-affinity binding between Protein G and Fc region of DBCO-modified IgG. This enabled the directional conjugation of DBCO-IgG with azide-modified nucleic acids via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry, achieving specific capture and enrichment of modified nucleic acids. Firstly, this method efficiently enriched two major DNA modifications, 8-oxo-7,8-dihydroguanine (8-oxo-dG) and 5-hydroxymethylcytosine (5hmC) in model DNA systems. Then, genome-wide distribution of 8-oxodG from human cells and tissues align with previously reports. Finally, employing ‘Click-IP-Seq’, we performed the first comprehensive analysis of 8-oxo-dG spatial distribution and associated biological functions in human colorectal carcinoma tissues. This technology provides a high-specificity and versatile enrichment platform for nucleic acid modifications, which is expected to promote the application of cancer molecular diagnosis.

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An experimentally generated peptide database increases the sensitivity in XL-MS of complex samples

Project description:The application of non-cleavable cross-linkers to complex samples in XL-MS workflows is limited by the n² problem, a quadratic expansion of the search space with increasing database size. Here, a peptide-focused approach is proposed, for which cross-linking peptide candidates are identified in a parallel experiment by using a thiol-cleavable cross-linker with equal reactivity. Samples are reduced and alkylated, thereby releasing cross-linked peptides with a variable modification on the initially cross-linked residue. Modified peptides are identified by database search and concatenated to a peptide database, which is finally used for the analysis of the sample cross-linked with a non-cleavable cross-linker. This way, the search space is dramatically reduced leading to a higher sensitivity, because there is less chance for random hits to false positive sequences. The approach was benchmarked on cross-linked purified protein complexes (20 S Proteasome, yeast PolII, and TFIIH), and in vivo cross-linked bacteria (Bacillus subtilis, Bacillus cereus) by comparing it to the conventional approach of searching against the protein sequences.

2020-03-26 | PXD016554 | Pride

Fast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides.

Project description:Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to sub-stochiometric reaction efficiencies, cross-linked peptides are usually low abundant. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose�� beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from ~100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2 �� 1:100. The number of detectable unique cross-links maintained high at ~100. The estimated enrichment efficiency was improved by factor 4 -5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 ��g cross-linked E. coli ribosome in a background of 100 ��g tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast and selective enrichment method for azide-tagged linkers will contribute to map protein-protein interactions, investigate protein architectures in more depth and help to understand complex biological processes.

2020-05-11 | PXD016963 | Pride

Cell Membrane Protein-centered Interactome Analysis Enabled by In-Vivo Cross-Linking Combined with Glycan-Directed Enrichment

Project description:a biocompatible, mass spectrometry (MS)-cleavable crosslinker, trehalose disuccinimidyl ester (TDS), was used to “freeze” interactions between membrane proteins and their partners with enhanced cross-linking and identification capabilities. This was coupled with a glycan-directed enrichment strategy based on protein-level hydrazine chemistry, allowing efficient isolation of crosslinked cell membrane proteins interacting proteins for further analysis.

2026-01-30 | PXD073835 |

A Glycosidic-Bond-Based Mass-Spectrometry-Cleavable Cross-linker Enables In Vivo Cross-linking for Protein Complex Analysis

Project description:Chemical cross-linking mass spectrometry (CXMS) has emerged as a powerful technology to analyze protein complex structure and interaction. However, the spectral fragmentation behavior and spectral data retrieval of cross-linked peptides are more complex than single peptides. In this study, we designed and synthesized a trehalose-based MS-cleavable cross-linker, Trehalose Disuccinimidyl Ester (TDS), which possesses a CID/HCD-cleavable glycosidic bond and has good bioorthogonality and amphipathicity. Using TDS, the cross-linked peptides were simplified into conventional single peptides via the selective cleavage between glycosidic and peptide bonds under individual MS collision energy, which enhances the matching degree and retrieval throughput of spectral identification. The deep coverage of the TDS method facilitated the accurate resolution of the structural dynamics of purified proteins with different physicochemical properties and yeast 26S proteasome complex. Additionally, the bioorthogonality and amphipathicity of TDS enabled the cross-linking reaction to occur in vivo without the introduction of any organic solvent. Through coinciding with this feature and MS-cleavable capacity, TDS provided us a high throughput snapshot of the structural architecture of protein complex in live cells. These results provide a promising TDS toolkit to study CXMS and decipher the protein conformations and interactions with high accuracy and easy portability for cross-linker design.

2023-12-06 | PXD047554 |

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