Design of CID-cleavable protein cross-linkers: identical mass modifications for simpler sequence analysis

Kandur, Wynne V.; Kao, Athit; Vellucci, Danielle; Huang, Lan; Rychnovsky, Scott D.

doi:10.1039/c5ob01410g

Cited by 14 publications

(11 citation statements)

References 31 publications

(105 reference statements)

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“…In addition, high-resolution mass spectrometry can identify direct sites of interaction between crosslinked amino acids, providing detailed spatial information for structural studies. [8][9] Recently, development of collision-induced dissociation (CID) cleavable cross-linkers [10][11][12][13][14] and improvements in algorithms for cross-link peptide spectral analysis [15][16][17][18][19] has enabled proteinprotein interaction mapping of complex proteomes, including worm, 20-21 bacteria, [21][22][23] and human [15][16][24][25] and various other organisms, 26 with hundreds-to-thousands of cross-links and protein-protein interactions. 16,21 While cross-linking of cell extracts provides direct control of the reaction to label specific protein complexes, [15][16][20][21] membrane-permeability of some cross-linking reagents enables cross-linking of protein complexes in live cells to define specific protein-protein interactions in situ.…”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Ser¹,

Cifani²,

Kentsis³

2018

Preprint

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“…For chemical cross-linking, the LRRK2 concentration was adjusted to 3 µM, and each Nb was added at a 2:1 molar ratio and incubated for 1 h at 4 °C. The cross-linking reaction was performed using the N-hydroxysuccinimide (NHS)-ester–based and collision-induced dissociation (CID)-cleavable reagent disuccinimidyl sulfoxide (Thermo Fisher Scientific) ( 60 ) at a molar excess of 60:1 (referred to the Nbs) and carried out for 30 min at room temperature. Proteins were precipitated by chloroform/methanol and subjected to tryptic proteolysis ( 61 ).…”

Section: Methodsmentioning

confidence: 99%

Nanobodies as allosteric modulators of Parkinson’s disease–associated LRRK2

Singh

Soliman

Guaitoli

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson’s disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.

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“…DSSO 19 , isotope-labeled (DMDSSO) 20 , and enrichable (Azide/Alkyne-A-DSBSO) [21][22] ), acidic residue-targeting (DHSO) 23 , and cysteine-reactive (BMSO) 24 cross-linkers. These MScleavable reagents contain symmetric MS-labile C-S bonds (adjacent to the sulfoxide group) that are selectively and preferentially fragmented prior to peptide backbone cleavage during CID 5,[19][20][22][23][24][25] . Such fragmentation has proven robust and predictable, occurring independently of cross-link types, peptide charges and sequences, thus enabling simplified and accurate identification of sulfoxide-containing cross-linked peptides by MS n analysis and conventional database searching tools.…”

Section: Introductionmentioning

confidence: 99%

Exploring Spacer Arm Structures for Designs of Asymmetric Sulfoxide-Containing MS-Cleavable Cross-Linkers

Novitsky

Cheng

et al. 2020

Anal. Chem.

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Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MScleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multi-stage mass spectrometry (MS n ). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of disuccinimidyl sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics and MS identification of crosslinked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS 2 -based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MS n analysis. This work has established a solid foundation for further development of sulfoxidecontaining MS-cleavable cross-linkers with new functionalities.

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Design of CID-cleavable protein cross-linkers: identical mass modifications for simpler sequence analysis

Cited by 14 publications

References 31 publications

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Nanobodies as allosteric modulators of Parkinson’s disease–associated LRRK2

Exploring Spacer Arm Structures for Designs of Asymmetric Sulfoxide-Containing MS-Cleavable Cross-Linkers

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