An integrated workflow for crosslinking mass spectrometry (original) (raw)
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SIM-XL: A powerful and user-friendly tool for peptide cross-linking analysis
Journal of Proteomics, 2015
Chemical cross-linking has emerged as a powerful approach for the structural characterization of proteins and protein complexes. However, the correct identification of covalently linked (cross-linked or XL) peptides analyzed by tandem mass spectrometry is still an open challenge. Here we present SIM-XL, a software tool that can analyze data generated through commonly used cross-linkers (e.g., BS3/DSS). Our software introduces a new paradigm for search-space reduction, which ultimately accounts for its increase in speed and sensitivity.
Efficient and robust proteome-wide approaches for cross-linking mass spectrometry
Nature Protocols, 2018
Crosslinking mass spectrometry (XL-MS) has received considerable interest due to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison to other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by use of bi-functional crosslinking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by mass spectrometry. XL-MS has received the required boost to tackle more complex samples with recent advances in crosslinking chemistry with MS-cleavable or reporter-based crosslinkers and faster, more sensitive and more versatile mass spectrometry platforms. This protocol provides a detailed description of our optimized conditions for a full proteome native protein preparation followed by crosslinking using the gas-phase cleavable crosslinking reagent DSSO. Following crosslinking, we demonstrate extensive sample fractionation and significantly simplified data analysis with XlinkX in Proteome Discoverer and subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within approximately 10 d including structural investigations.
Using SIM-XL to identify and annotate cross-linked peptides analyzed by mass spectrometry
Protocol Exchange, 2015
State-of-the-art structural mass spectrometry-based proteomics is often accomplished by using crosslinkers to covalently bind two or more amino-acid groups. This strategy is complementary to classical structural biology and therefore broadens the toolset for analyzing protein and protein-protein complex structures. One of the greatest challenges in identifying cross-linked peptides in complex protein mixtures is computationally dealing with the large search space, which grows quadratically with each peptide included in the sequence database. The **Spectrum Identi cation Machine for Cross-Linked Peptides \ (SIM-XL)** software uses an algorithm that overcomes this limitation by capitalizing on experimental features that allow it to effectively address the massive combinatorial problem at hand, and presents the results in a user-friendly manner. Thus, SIM-XL is recommended for studies dealing with protein structure and protein-protein interaction in either simple or complex protein mixtures. SIM-XL also allows the sharing of results through PRIDE by exporting them in the mzIdentML format.
Analytical Chemistry, 2009
Chemical crosslinking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of crosslinked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact crosslinkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under condition where the native complex structure is stable. In this study, we present a novel compact crosslinker that contains two distinct features: 1) an alkyne tag and 2) a small molecule detection tag (NO 2 -) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the crosslinked peptide after proteolytic cleavage after coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO 2 -moiety provides a secondary means of detecting crosslinked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this crosslinker, which we termed CLIP (Clickenabled Linker for Interacting Proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for crosslinked ubiquitin in highly complex E. coli cell lysates. Sequential CID-MS/ MS and ETD-MS/MS of inter-crosslinked peptides (two peptides connected with a crosslinker) are also demonstrated for improved automated identification of crosslinked peptides.
Large Scale Chemical Cross-linking Mass Spectrometry Perspectives
Journal of proteomics & bioinformatics, 2013
The spectacular heterogeneity of a complex protein mixture from biological samples becomes even more difficult to tackle when one's attention is shifted towards different protein complex topologies, transient interactions, or localization of PPIs. Meticulous protein-by-protein affinity pull-downs and yeast-two-hybrid screens are the two approaches currently used to decipher proteome-wide interaction networks. Another method is to employ chemical cross-linking, which gives not only identities of interactors, but could also provide information on the sites of interactions and interaction interfaces. Despite significant advances in mass spectrometry instrumentation over the last decade, mapping Protein-Protein Interactions (PPIs) using chemical cross-linking remains time consuming and requires substantial expertise, even in the simplest of systems. While robust methodologies and software exist for the analysis of binary PPIs and also for the single protein structure refinement usin...
Analytical chemistry, 2016
Chemical cross-linking and mass spectrometry are now widely used to analyze large-scale protein-protein interactions. The major challenge in cross-linking approaches is the complexity of the mass spectrometric data. New approaches are required that can identify cross-linked peptides with high-confidence and establish a user-friendly analysis protocol for the biomedical scientific community. Here, we introduce a novel cross-linker that can be selectively cleaved in the gas phase using two differential tandem mass-spectrometric fragmentation methods, such as collision-induced or electron transfer dissociation (CID and ETD). This technique produces two signature mass spectra of the same cross-linked peptide, thereby producing high confidence in identifying the sites of interaction. Further tandem mass spectrometry can also give additional confidence on the peptide sequences. We demonstrate a proof-of-concept for this method using standard peptides and proteins. Peptides and proteins we...
CLPM: a cross-linked peptide mapping algorithm for mass spectrometric analysis
2005
Background: Protein-protein, protein-DNA and protein-RNA interactions are of central importance in biological systems. Quadrapole Time-of-flight (Q-TOF) mass spectrometry is a sensitive, promising tool for studying these interactions. Combining this technique with chemical crosslinking, it is possible to identify the sites of interactions within these complexes. Due to the complexities of the mass spectrometric data of crosslinked proteins, new software is required to analyze the resulting products of these studies. Result: We designed a Cross-Linked Peptide Mapping (CLPM) algorithm which takes advantage of all of the information available in the experiment including the amino acid sequence from each protein, the identity of the crosslinker, the identity of the digesting enzyme, the level of missed cleavage, and possible chemical modifications. The algorithm does in silico digestion and crosslinking, calculates all possible mass values and matches the theoretical data to the actual experimental data provided by the mass spectrometry analysis to identify the crosslinked peptides. Conclusion: Identifying peptides by their masses can be an efficient starting point for direct sequence confirmation. The CLPM algorithm provides a powerful tool in identifying these potential interaction sites in combination with chemical crosslinking and mass spectrometry. Through this cost-effective approach, subsequent efforts can quickly focus attention on investigating these specific interaction sites.
Molecular & Cellular Proteomics, 2011
Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS 3 analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS 3 ) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes. Molecular &
A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry
PloS one, 2016
Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and s...
Analytical Chemistry, 2016
Cross-linking combined with mass spectrometry (MS) has evolved as an alternative strategy in structural biology for characterizing three-dimensional structures of protein assemblies and for mapping protein-protein interactions. Here, we describe an integrated workflow for an automated identification of cross-linked products that is based on the use of an MS/MS cleavable cross-linker (BuUrBu) generating characteristic doublet patterns upon fragmentation. We evaluate different fragmentation methods available on an Orbitrap Fusion mass spectrometer for three proteins and an E. coli cell lysate. An updated version of the dedicated software tool MeroX was employed for a fully automated identification of crosslinks. The strength of our cleavable cross-linker is that characteristic patterns of the crosslinker as well as backbone fragments of the connected peptides are already observed at the MS/MS level, eliminating the need for conducting MS 3 or sequential CID (collision-induced dissociation)-and ETD (electron transfer dissociation)-MS/MS experiments. This makes our strategy applicable to a broad range of mass spectrometers with MS/MS capabilities. For purified proteins and protein complexes our workflow using CID-MS/MS acquisition performs with high confidence, scoring cross-links at 0.5% false discovery rate (FDR). The cross-links provide structural insights into the intrinsically disordered tetrameric tumor suppressor protein p53. As a time-consuming manual inspection of cross-linking data is not required, our workflow will pave the way for making the cross-linking/MS approach a routine technique for structural proteomics studies.