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      Quantitative cross-linking/mass spectrometry to elucidate structural changes in proteins and their complexes

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      Nature Protocols

      Springer Nature

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          Abstract

          Quantitative cross-linking/mass spectrometry (QCLMS/QXL-MS) probes structural changes of proteins in solution. This method has revealed induced conformational changes, composition shifts in conformational ensembles and changes in protein interactions. It illuminates different structural states of proteins or protein complexes by comparing which residue pairs can be cross-linked in these states. Cross-links provide information about structural changes that may be inaccessible by alternative technologies. Small local conformational changes affect relative abundances of nearby cross-links, whereas larger conformational changes cause new cross-links to be formed. Distinguishing between minor and major changes requires a robust analysis based on carefully selected replicates and, when using isotope-labeled cross-linkers, replicated analysis with a permutated isotope-labeling scheme. A label-free workflow allows for application of a wide range of cross-linking chemistries and enables parallel comparison of multiple conformations. In this protocol, we demonstrate both label-free and isotope-labeled cross-linker-based workflows using the cross-linker bis(sulfosuccinimidyl)suberate (BS3). The software XiSearch, developed by our group, is used to identify cross-linked residue pairs, although the workflow is not limited to this search software. The open-access software Skyline is used for automated quantitation. Note that additional manual correction greatly enhances quantitation accuracy. The protocol has been applied to purified multi-protein complexes but is not necessarily limited to that level of sample complexity. Optimizing the cross-linker/protein ratio and fractionating peptides increase the data density of quantified cross-links, and thus the resolution of QCLMS. The entire procedure takes ~1-3 weeks.

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          Most cited references 54

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          MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.

          Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
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            Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.

            Skyline is a Windows client application for targeted proteomics method creation and quantitative data analysis. It is open source and freely available for academic and commercial use. The Skyline user interface simplifies the development of mass spectrometer methods and the analysis of data from targeted proteomics experiments performed using selected reaction monitoring (SRM). Skyline supports using and creating MS/MS spectral libraries from a wide variety of sources to choose SRM filters and verify results based on previously observed ion trap data. Skyline exports transition lists to and imports the native output files from Agilent, Applied Biosystems, Thermo Fisher Scientific and Waters triple quadrupole instruments, seamlessly connecting mass spectrometer output back to the experimental design document. The fast and compact Skyline file format is easily shared, even for experiments requiring many sample injections. A rich array of graphs displays results and provides powerful tools for inspecting data integrity as data are acquired, helping instrument operators to identify problems early. The Skyline dynamic report designer exports tabular data from the Skyline document model for in-depth analysis with common statistical tools. Single-click, self-updating web installation is available at http://proteome.gs.washington.edu/software/skyline. This web site also provides access to instructional videos, a support board, an issues list and a link to the source code project.
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              Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.

              Mass spectrometry (MS)-based proteomics measures peptides derived from proteins by proteolytic cleavage. Before performing the analysis by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS), nanoelectrospray-MS/MS (NanoES-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS), the peptide mixtures need to be cleaned, concentrated and often selectively enriched or pre-fractionated, for which we employ simple, self-made and extremely economical stop-and-go-extraction tips (StageTips). StageTips are ordinary pipette tips containing very small disks made of beads with reversed phase, cation-exchange or anion-exchange surfaces embedded in a Teflon mesh. The fixed nature of the beads allows flexible combination of disks with different surfaces to obtain multi-functional tips. Disks containing different surface functionalities and loose beads such as titania and zirconia for phosphopeptide enrichment can be combined. Incorporation into an automated workflow has also been demonstrated. Desalting and concentration takes approximately 5 min while fractionation or enrichment takes approximately 30 min.
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                Author and article information

                Journal
                Nature Protocols
                Nat Protoc
                Springer Nature
                1754-2189
                1750-2799
                December 17 2018
                Article
                10.1038/s41596-018-0089-3
                30559374
                © 2018

                http://www.springer.com/tdm

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