An Isotopically Coded CID-cleavable Biotinylated Cross-linker for Structural Proteomics

Petrotchenko, Evgeniy V.; Serpa, Jason J.; Borchers, Christoph H.

doi:10.1074/mcp.m110.001420

Cited by 120 publications

(124 citation statements)

References 18 publications

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“…For example, xIP-MS would benefit directly from novel ultrahigh-affinity GFP nanobody dimers, as reported recently (62). Also, developments in enrichable or cleavable cross-linkers could facilitate higher cross-link identification rates (16,20,63). Therefore, xIP-MS can also be considered as an experimental platform for rapid optimization of novel cross-linking strategies, thereby extending the toolbox of structural proteomics technologies.…”

Section: Discussionmentioning

confidence: 99%

Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification

Makowski

Willems

Jansen

et al. 2016

Molecular & Cellular Proteomics

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In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS using the structurally well-characterized phosphoribosyl pyrophosphate synthetase complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCM complexes, and we provide novel structural and biological insights into their architectures and molecular function. Of note, we use xIP-MS to perform topological studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for interrogating chromatin-associated protein complex architectures. Molecular & Cellular Proteomics 15: 10.1074/mcp.M115.053082, 854-865, 2016.The structural basis of specific protein-protein interactions and higher-order protein complex multimerization crucially informs our understanding of many molecular and cellular processes. AP-MS-based strategies have been a major player in the elucidation of specific protein-protein interactions and core protein complexes (1-3). However, although quantitative AP-MS/MS experiments identify specific interactors for any given bait, they cannot differentiate between direct and indirect interactors or provide structural or topological information on protein complex assemblies. Furthermore, traditional structural methodologies often fail with large or dynamic protein complexes including chromatin remodeling or chromatin associated protein complexes. Cross-linking mass spectrometry (XL-MS), 1 therefore, has emerged as a powerful technique for analyzing protein complex architectures through direct observation of subcomplex interfaces at the peptide level (4 -6). Landmark studies used XL-MS to reveal the architectures of the chromatin remodeling complexes Ino80 and SWR1 (7,8), and other chromatin-associated complexes have been interrogated using traditional XL-MS workflows (9 -14).In general, cross-linked peptides represent a small fraction of the total pool of peptides measured in an MS/MS analysis. Therefore, traditional XL-MS workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows to facilitate the detection of low abundance cross-linked peptides from...

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Section: Discussionmentioning

confidence: 99%

Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification

Makowski

Willems

Jansen

et al. 2016

Molecular & Cellular Proteomics

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“…We expect that recent strategies to analyze protein complexes using quantitative MS will further improve our understanding of such molecular clusters. [187][188][189][190][191] In addition, quantitative MS-based profiling methods for cell organelles can provide information about the spatial distribution of proteins 192,193 and reveal dynamic changes in this distribution. Given the role of circulating platelets as sentinels of vascular integrity, proteomic analyses and PTM signatures could lead to new approaches for diagnosis and treatment of platelet disorders.…”

Section: Future Directions Of Platelet Proteomicsmentioning

confidence: 99%

What Can Proteomics Tell Us About Platelets?

Burkhart

Gambaryan

Watson

et al. 2014

Circulation Research

102

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More than 130 years ago, it was recognized that platelets are key mediators of hemostasis. Nowadays, it is established that platelets participate in additional physiological processes and contribute to the genesis and progression of cardiovascular diseases. Recent data indicate that the platelet proteome, defined as the complete set of expressed proteins, comprises >5000 proteins and is highly similar between different healthy individuals. Owing to their anucleate nature, platelets have limited protein synthesis. By implication, in patients experiencing platelet disorders, platelet (dys)function is almost completely attributable to alterations in protein expression and dynamic differences in post-translational modifications. Modern platelet proteomics approaches can reveal (1) quantitative changes in the abundance of thousands of proteins, (2) post-translational modifications, (3) protein–protein interactions, and (4) protein localization, while requiring only small blood donations in the range of a few milliliters. Consequently, platelet proteomics will represent an invaluable tool for characterizing the fundamental processes that affect platelet homeostasis and thus determine the roles of platelets in health and disease. In this article we provide a critical overview on the achievements, the current possibilities, and the future perspectives of platelet proteomics to study patients experiencing cardiovascular, inflammatory, and bleeding disorders.

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“…Model Cross-linked Peptide-The model peptide Ac-TRTEST-DIKRASSREADYLINKER (Creative Molecules Inc., Victoria, Canada) was cross-linked with an equimolar amount of CBDPS-H 8 /D 8 reagent (cyanurbiotindipropionylsuccinimide; Creative Molecules Inc.), as previously described (10). The pH of the mixture was adjusted to 8.0 -8.5 by the addition of 0.2 M Na 2 HPO 4 .…”

Section: Methodsmentioning

confidence: 99%

“…1). CBDPS is homo-bifunctional NHS-based amine-reactive cross-linker, possessing additional features useful for mass spectomeric analyses, such as isotopic coding, CID cleavage, and affinity purification (10). Previous studies have shown that intrapeptide cross-links are generated by cross-linking this peptide with CBDPS.…”

Section: Digestion Of the Cross-linked Modelmentioning

confidence: 99%

Use of Proteinase K Nonspecific Digestion for Selective and Comprehensive Identification of Interpeptide Cross-links: Application to Prion Proteins

Petrotchenko

Serpa

Hardie

et al. 2012

Molecular & Cellular Proteomics

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Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight crosslinked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP C ) and oligomeric form of prion protein (PrP␤). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the crosslinked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP Structural proteomics, which combines protein chemistry methods with modern mass spectrometry techniques for protein and peptides, is an emerging technology in structural biology. One of the tools for modern structural proteomics is chemical cross-linking combined with mass spectrometry (1-5). Cross-linking analysis provides the distance between two cross-linked amino acid residues, whereas mass spectrometry provides information on which residues are connected. The basis of this method is a chemical reaction of the crosslinking reagent with functional groups on a protein, leading to the formation of a covalent bond between each end of the reagent and the protein. The distance between two crosslinked sites is determined by the length of the spacer in the cross-linking reagent. Thus, identification of the cross-linked sites on a protein or a protein complex provides spatial information and distance constraints for the two amino acid residues that are connected by the cross-linker.The use of chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics (4, 6 -8). In recent years, we and others have been developing an array of cross-linking and modification reagents, software, and methods specifically designed for protein cross-linking experiments combined with MS analysis (9 -18). For mass spectrometric determina...

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An Isotopically Coded CID-cleavable Biotinylated Cross-linker for Structural Proteomics

Cited by 120 publications

References 18 publications

Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification

Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification

What Can Proteomics Tell Us About Platelets?

Use of Proteinase K Nonspecific Digestion for Selective and Comprehensive Identification of Interpeptide Cross-links: Application to Prion Proteins

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