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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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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

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.

2019-11-04 | GSE122542 | GEO

Genome-wide maps of GlcNAc bound proteins and Pho ChIP-seq in Drosophila S2 cells and pupae.

Project description:We developed a strategy to map GlcNAc modified proteins based on a chemical chromatin precipitation strategy. Drosophila S2 cells, as well as wild type (wt) and sxc null pupae, were fed with 100 uM Ac4GalNAz and the resulting DNA was cross-linked, sonicated, and purified. DNA strands cross-linked to GlcNAz proteins were isolated by congugating with alkyn-biotin by Staudinger ligation followed by streptavidin purification. The resulting DNA was constructed into libraries for sequencing. To asses the robustness of our strategy, we compared GalNAz ChIP-seq results in S2 cells with two other GlcNAc ChIP-seq strategies, using a mutant β-1,4-galactosyltransferase (GalT) and the lecting wheat germ agglutinin (WGA). Briefly, GalT was incubated with cross-linked, sonicated and purified DNA along with UDP-GalNAz. Ligation to biotin with click chemistry followed by streptavidin purification resulted in library ready material. For WGA and Pho ChIP-seq, cross-linked and purified DNA was incubated with pho antibody or WGA resin and purified followed by library preperation.

2017-07-18 | GSE84502 | GEO

A simplified in vivo cross-linking mass spectrometry strategy for proteome-wide studies.

Project description:Chemical cross-linking (XL) coupled to mass spectrometry (MS) has become a powerful approach to probe the structure of protein assemblies. Although most of the applications concerned purified complexes, latest developments focus on large-scale in vivo studies. Pushing in this direction, we describe a new and simplified in vivo XL-MS workflow to study the cellular interactome of living bacterial cells. It is based on in vivo labeling and involves a one-step enrichment by click-chemistry on a solid support. Our approach shows an impressive efficiency on Neisseria meningitidis, leading to the identification of about 1,800 cross-links in a single LC-MS/MS run using a benchtop high-resolution Orbitrap mass spectrometer. Highly dynamic multiprotein complexes were successfully captured and characterized in all bacterial compartments, showing the great potential and precision of our large-scale approach. Our workflow paves new avenues for the deep in vivo analysis of cellular interactomes.

2021-02-25 | PXD021553 | Pride

Open search strategy for inferring the masses of cross-link adducts

Project description:Development of new reagents for protein cross-linking is continually ongoing. The chemical formulas of the linker adducts formed by these reagents are usually deduced from expert knowledge, and then validated by mass spectrometry. Clearly, it would have been more rigorous if the adduct masses are inferred directly from the data without any prior assumption on the chemistries. Unfortunately, the analysis tools that are currently available to detect chemical modifications on linear peptides are not applicable to the case of two cross-linked peptides. Here we show that an adaptation of the open search strategy that works on linear peptides can be used to study cross-link modifications between pairs of peptides. We benchmark our approach by correctly inferring the linker masses of two well-known reagents - DSS and formaldehyde - to accuracies of a few parts per million. We then investigate the cross-linking chemistries of two poorly characterized reagents - EMCS and glutaraldehyde. In the case of EMCS, we find that the expected cross-linking chemistry is accompanied by a competing chemistry that targets other amino acid types. In the case of glutaraldehyde, we find that the chemical formula of the dominant linker is C5H4, which indicates a ringed aromatic structure. These results demonstrate that, at very little effort, our approach can yield non-trivial insights to facilitate the studies of new cross-linkers.

2021-09-09 | PXD020704 | Pride

In planta chemical crosslinking and mass spectrometry analysis of protein structure and interaction in Arabidopsis

Project description:Mass spectrometry analysis in combination with the site-specific chemical cross-linking has emerged as a powerful method in study of three-dimensional structure of protein complex and in mapping of protein-protein interactions (PPIs). Even though in vitro cross-linking experiments have been widely applied to investigate the specific interactions of a bait protein and its targets, the measurement of in vivo protein tertiary structure and PPIs has been problematic and strenuous due to the dynamic nature of the biological systems and a lower number of cross-linked peptides that can be isolated via MudPIT (Multidimensional Protein Identification Technology) for mass spectrometry analysis. Using Arabidopsis thaliana as a model multicellular eukaryotic organism, we have attempted to develop an improved in vivo chemical cross-linking and mass spectrometry (or IPXL-MS) workflow, which aims at optimizing the in vivo cross-linking conditions, establishing of a MudPIT procedure for enrichment of cross-linked peptides, and developing an integrated software program to identify the in planta chemical cross-linked peptides, by which three pairs of in vivo cross-linked peptides of high-confidence has been identified twice from two independent biological replicates. This work has demarked a beginning of alternative proteomic approach in study of in vivo protein tertiary structure and PPIs in higher plants. This in vivo cross-linking approach may be applied into other model multicellular organisms, such as mouse, for molecular systems biological research.

2016-06-28 | PXD003658 | Pride

Reverse-phase liquid chromatography (RPLC) in Combination with Tip-Based Strong Cation Exchange (SCX-Tip) Fractionation, ReST, an Alternative Approach for Cross-linked Peptides analysis

Project description:Chemical cross-linking in combination with mass spectrometry (XL-MS) has emerged as a useful method for structural elucidation of proteins and protein complexes. Efficient enrichment procedures are necessary to analyze cross-linked products due to their relatively low abundance. Currently, strong cation exchange chromatography (SCX), size exclusion chromatography (SEC), and affinity tag-based enrichment are among the widely used enrichment strategies. Herein, we present a two-dimensional enrichment strategy combining basic RPLC (bRPLC) fractionation and tip-based SCX (SCX-Tip) enrichment, termed ReST method, for the characterization of cross-linked peptides. We revealed the unbiased separation effects of the bRPLC in the cross-linked peptide fractionation. We optimized the enrichment conditions of SCX-Tip for cross-linked peptides using well-designed cross-linked peptides. Taking advantage of the high resolution of bRPLC fractionation and the high enrichment efficiency of SCX-Tip, we were able to identify 43.6% more cross-links than the conventional SCX approach. The presented ReST approach is an effective fractionation method for XL-MS analysis, and could be beneficial for protoeme-scale protein-protein interaction studies. Moreover, more stringent data filtering was proposed in order to achieve the high confidence of cross-linked peptide identification.

2022-06-16 | PXD023817 | Pride

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

eXL-MS: An enhanced Cross-Linking Mass Spectrometry Workflow to Study Protein Complexes.

Project description:The analysis of proteins and protein complexes by cross-linking mass spectrometry (XL-MS) has expanded in the last dec-ade. However, mostly used approaches suffer important limitations in term of efficiency and sensitivity. We describe here a new workflow based on the advanced use of the trifunctional cross-linker NNP9. NNP9 carries an azido group allowing the quantitative and selective introduction of a biotin molecule into cross-linked proteins. The incorporation is performed by click-chemistry using an adapted version of the enhanced filter-aided sample preparation (eFASP) protocol. This protocol, based on the use of a molecular filter, allows a very high recovery of peptides after enzymatic digestion and complete re-moval of contaminants. This in turn offers the possibility to analyze very large membrane proteins solubilized in detergent. After trypsin digestion, biotinylated peptides can be easily enriched on monoavidin beads and analyzed by LC-MS/MS. The whole workflow was developed on creatine kinase in presence of detergent. It led to a drastic improvement in the number of identified cross-linked peptides (407 vs 81), compared to the conventional approach using a gel-based separation. One great advantage of our eXL-MS workflow is its high efficiency, allowing the analysis of very low amount of material (15 mi-crograms). We also demonstrate that Higher-Energy Collision Dissociation (HCD) outperforms Electron-Transfer/Higher-Energy Collision Dissociation (EThcD) in term of number of cross-linked peptides identified, but EThcD leads to better se-quence coverage than HCD and thus easier localization of cross-linking sites.

2021-08-11 | PXD010422 | Pride

Poly (A) Click-seq for U937 control and PMA-treated (6h and 24h) cells

Project description:Poly(A)-ClickSeq: click-chemistry for next-generation 3'-end sequencing without RNA enrichment or fragmentation enabling simultaneous genomewide poly(A)-site identification and differential expression analysis

2023-01-12 | GSE169140 | GEO

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