Histone Interaction Landscapes Visualized by Crosslinking Mass Spectrometry in Intact Cell Nuclei

Fasci, Domenico; Ingen, Hugo van; Scheltema, Richard A.; Heck, Albert J. R.

doi:10.1074/mcp.ra118.000924

Cited by 119 publications

(128 citation statements)

References 83 publications

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“…In particular, we observed numerous cross-links involving histone H2B K5 and histone H3 K4 and K18, which were also found in a recent study. 61 Notably, using labeling of protein complexes in situ, we observed numerous previously unrecognized interactions. For example, we found numerous cross-links between the HMGN1 and HMGN2 proteins with histone H2B and histone H1.2 ( Figure 5D & E).…”

Section: Analysis Of the Cross-linked Chromatin Fraction Identifiedmentioning

confidence: 96%

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Ser¹,

Cifani²,

Kentsis³

2018

Preprint

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Recent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS).Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the density and coverage of protein-protein interaction maps can be significantly improved through the use of multiple proteases. In addition, the use of sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching.Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems. 5 Experimental Section Reagents. Disuccinimidyl sulfoxide (DSSO), formic acid, dimethyl sulfoxide (DMSO), and LC-MS grade solvents were obtained from Thermo Scientific. Bovine serum albumin (BSA), (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride, magnesium chloride, potassium chloride, sucrose, ethylenediaminetetraacetic acid (EDTA), dithiothreitol, guanidinium hydrochloride, Staphylococcus aureus micrococcal nuclease, iodoacetamide, ammonium bicarbonate and formic acid were obtained from Millipore Sigma. Protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and pepstatin were obtatined from Santa Cruz, bestatin from Alfa Aesar and leupeptin from EMD Millipore. Trypsin and GluC proteases were obtained from Promega. Chymotrypsin protease was obtained from Pierce Thermo. LysC was obtained from Wako Pure Chemical Industries.Cell culture and protein isolation. HEK293T cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin and streptomycin. HEK293T cells (50 million) were sedimented by centrifugation at 500 g and lysed in 500 µl 6M guanidine hydrochloride in 100 mM ammonium bicarbonate buffer, pH 8. The lysate was sonicated (E210 adaptive focused acoustic sonicator, Covaris) for 5 minutes at 4°C and homogenized by passing through a 27G needle. The lysate was then clarified by centrifugation at 18,000 g for 10 min at 4°C. Protein concentration of clarified lysates was determined using the bicinchoninic acid (BCA) assay, according to manufacturer's instructions (Thermo Scientific).Preparation of cross-linked bovine serum albumin and peptide purification. 50 µg of BSA was solubilized in 50 µl HEPES buffer (20 mM HE...

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Section: Analysis Of the Cross-linked Chromatin Fraction Identifiedmentioning

confidence: 96%

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Ser¹,

Cifani²,

Kentsis³

2018

Preprint

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“…One approach would be to combine crosslinking-mass spectrometry (XL-MS) with any of the chromatin-domain enrichment procedures described above. While potentially biochemically challenging, XL-MS has readily been shown to be applicable to intact nuclei (84), providing an important step towards obtaining crosslinking maps of enriched chromatin fragments. If XL-MS could be adapted for application in chromatin enrichment workflows, this will have as extra benefit as it allows to distinguish direct from indirect protein interactions, which is currently not yet possible with the described chromatin (domain) enrichment strategies.…”

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%

Chromatin Proteomics to Study Epigenetics — Challenges and Opportunities

Mierlo

Vermeulen

2021

Molecular & Cellular Proteomics

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Regulation of gene expression is essential for the functioning of all eukaryotic organisms. Understanding gene expression regulation requires determining which proteins interact with regulatory elements in chromatin. Mass spectrometry-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation, as it allows studying protein function and protein complex formation in their in vivo chromatin-bound context. Total chromatin isolated from cells can be directly analysed using mass spectrometry or further fractionated into transcriptionally active and inactive chromatin prior to MS-based analysis. Newly formed chromatin that is assembled during DNA replication can also be specifically isolated and analysed. Furthermore, capturing specific chromatin domains facilitates the identification of previously unknown transcription factors interacting with these domains. Finally, in recent years, advances have been made towards identifying proteins that interact with a single genomic locus of interest. In this review, we highlight the power of chromatin proteomics approaches and how these provide complementary alternatives compared to conventional affinity purification methods. Furthermore, we discuss the biochemical challenges that should be addressed to consolidate and expand the role of chromatin proteomics as a key technology in the context of gene expression regulation and epigenetics research in health and disease.

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“…In this dataset, 158 of the 307 crosslinks (identified by 314 spectra) are solely on the 80S ribosome, a 50% higher number than reported in previous reports. [7,45] Of the 158 crosslinks, 71 crosslinks could be mapped on the cryo-EM structure of the 80S human ribosome (PDB:4v6x), the others are predominantly in regions not resolved in the structural model of the ribosome. Of the mapped crosslinks, 90% are within the defined distance constraint of 20Å (Supplementary Note 12).…”

Section: Enrichment Efficiencymentioning

confidence: 99%

“…For previous efforts, extensive fractionation was required to get similar numbers (e.g. for the XL-MS study on the nucleus, 86 measurements, totaling 172 hrs of measurement time was needed to extract 87 crosslinks on the ribosome [45] ), clearly demonstrating the sensitivity and efficiency of PhoX.…”

Section: Enrichment Efficiencymentioning

confidence: 99%

PhoX – an IMAC-enrichable Crosslinking Reagent

Steigenberger

Pieters

Heck

et al. 2019

Preprint

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Chemical crosslinking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, sub-stoichiometric reaction efficiencies render routine detection of crosslinked peptides problematic. Here we present a new tri-functional crosslinking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the crosslinked peptides amenable to the well-established IMAC enrichment. The handle allows for 300x enrichment efficiency and 97% specificity, dramatically reducing measurement time and improving data quality. We exemplify the approach on various model proteins and protein complexes, e.g. resulting in a structural model of the LRP1/RAP complex. PhoX is also applicable to whole cell lysates. When focusing the database search on ribosomal proteins, our first attempt resulted in 355 crosslinks, out-performing current efforts in less measurement time.

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Histone Interaction Landscapes Visualized by Crosslinking Mass Spectrometry in Intact Cell Nuclei

Cited by 119 publications

References 83 publications

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Chromatin Proteomics to Study Epigenetics — Challenges and Opportunities

PhoX – an IMAC-enrichable Crosslinking Reagent

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