A Modular Cross-Linking Approach for Exploring Protein Interactions

Trester-Zedlitz, Michelle; Kamada, Katsuhiko; Burley, S.K.; Fenyö, David; Chait, Brian T.; Muir, Tom W.

doi:10.1021/ja026917a

Cited by 185 publications

(174 citation statements)

References 24 publications

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“…In order to increase the yield of high quality assignments of cross-linked peptides, they will need to be purified directly. There have been suggestions for enrichment strategies based on affinity tagged cross-linkers, which, however, are not available in isotopically coded form yet or have isotopic signatures too small for electrospray ionization 14,15 . Having shown that high throughput identification of cross-linked peptides is now feasible it should spark interest to invest in advanced enrichment strategies for crosslinked peptides.…”

Section: Discussionmentioning

confidence: 99%

Identification of cross-linked peptides from large sequence databases

et al. 2008

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We describe a method to identify cross-linked peptides from complex samples and large protein sequence databases. The advance was achieved by combining isotopically tagged cross-linkers, chromatographic enrichment, targeted proteomics, and a novel search engine called xQuest. This software reduces the search space by an upstream candidatepeptide search before the recombination step; we show that xQuest can identify cross-linked peptides from a total E. coli lysate with an unrestricted database search.

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Section: Discussionmentioning

confidence: 99%

Identification of cross-linked peptides from large sequence databases

et al. 2008

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“…However, due to multiple major technical obstacles it had been impossible until recently to directly and reliably identify crosslinked peptides from protein complexes by MS (see [1][2][3][4] for recent reviews). After early work on individual proteins [5][6][7] and protein complexes [8,9], notably by the Sinz and Rappsilber groups, Aebersold and coworkers introduced the first robust general workflow by optimizing wet-lab protocols and the development of the publicly available xQuest/xProphet open-source software suite for the analysis and validation of crosslinks from large protein complexes by MS [10][11][12].Over the past few years the field has seen significant progress and several methods to enrich crosslinks [13][14][15], various crosslinking chemistries [9,10,[15][16][17][18][19][20][21][22], and the introduction of multiple detection and identification strategies [9][10][11][23][24][25][26][27][28]. Statistical models that differentiate true from false identifications have also been developed [10].…”

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confidence: 99%

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Leitner

Faini

Stengel

et al. 2016

Trends in Biochemical Sciences

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In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryoelectron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges. Chemical Crosslinking as a Tool for Structural BiologyStructural biology makes use of many different techniques to elucidate the 3D structures of proteins and protein complexes. While high-resolution structures have traditionally been obtained by X-ray crystallography, cryo-EM is increasingly able to also generate (near) atomic-resolution models. In recent years, techniques and applications of MS have also rapidly progressed. Earlier studies were largely focused on the large-scale identification and quantification of proteins, whereas recent methods also support queries into the composition, stoichiometry, and spatial arrangement of subunits in a complex. These developments have now further progressed toward generating information that contributes, as part of hybrid structural strategies, to the structure elucidation of large molecular assemblies including protein complexes that perform essential processes in the cell. XL-MS is a particularly powerful mass spectrometric technique in this respect, because it provides several layers of information. Identifying protein-protein contacts through XL-MS confirms physical proximity between subunits because the proteins must be close enough in space to be crosslinked. Localizing the side chains that are connected restricts this proximity to certain regions (e.g., domains or even single helices or loops). Finally, the structure of the connected side chains and the crosslinker moiety impart a distance restraint that can be used for molecular modeling purposes because an upper Trends Chemical crosslinking followed by the mass spectrometric analysis of cross linked peptides (XL MS) identifies con tact sites between residues within a single or between multiple proteins.The application of XL MS to many bio logically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2 3 years.Crosslinking data are useful in integra tive modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL MS has been shown to be particularly powerful in combination with 3D cryo electron microscopy. Sciences ; 41 (2016), 1. -S. 20-32 https://dx.doi.org/10.1016...

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“…Other biotinylated crosslinkers could be synthesized to accommodate a wider variety of proteins and protein complexes. A biotinylated homobifunctional crosslinker with two NHS groups, both reactive toward primary amines on proteins [21], is one such possibility; such a reagent would reduce the non-specificity introduced by the arylazide group of sulfo-SBED. Preliminary evidence suggests that tandem mass spectrometry experiments, such as MS-MS or MS 3 , can identify the two peptides in a crosslinked pair [5,10,28].…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, a separation method for enriching the analyte mixture in the crosslinked species would be useful for reducing levels of non-crosslinked peptides while simultaneously increasing the concentration of crosslinked species in the analyte mixture. An affinity separation provides such an advantage, as demonstrated by Trester-Zedlitz et al [21]. The biotin-avidin interaction is a logical choice for this affinity separation; a biotin-labeled crosslinker can be extracted, along with its attached peptide(s), from a complex peptide mixture using an immobilized avidin separation medium.…”

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confidence: 99%