Identification Of Cross-links Research Articles

Discovery and Characterization of an Acid-Labile Serine-Lysine Cross-Link in Antibody High-Molecular-Weight Species Using a Multipronged Mass Spectrometry Approach.

Characterizing the cross-links responsible for the covalent high-molecular-weight (HMW) species in therapeutic monoclonal antibodies (mAbs) is of great importance as it not only provides a framework for risk assessment but also offers insights for process improvement. However, owing to the complexity and low abundance, identification of novel and unknown cross-links in mAb products can be very challenging. Here, applying a multipronged MS-based approach, we report the discovery of a novel covalent cross-link formed via an imine bond between lysine and serine residues. In particular, this Ser-Lys cross-link was found to be acid-labile and can be easily overlooked by conventional LC-MS techniques operated at low pH. It is worth noting that although imine-based cross-link has been previously reported in collagen protein cross-linking, this is the first time that a Ser-Lys cross-link has been found in a mAb product that contributes to covalent HMW species formation.

Determination of HER2 binding domain in antigen-antibody complexes based on chemical crosslinking mass spectrometry

Chemical crosslinking (XL) of non-covalent antigen-antibody complexes followed by mass spectrometric identification (MS) of inter-protein crosslinks can provide spatial constraints between relevant residues, which are valuable structural information associated with the molecular binding interface. To highlight the potential of XL/MS in the biopharmaceutical industry, we herein developed and validated an XL/MS workflow that employed a zero-length linker, 1,1′‑carbonyldiimidazole (CDI), and a widely used medium-length linker, disuccinimidyl sulfoxide (DSSO), for fast, accurate determination of antigen domains targeted by therapeutic antibodies. To avoid false identification, system suitability samples and negative samples were designed for all experiments, and all tandem mass spectra were manually examined. To validate the proposed XL/MS workflow, two complexes involving human epidermal growth factor receptor 2 Fc fusion protein (HER2Fc) with known crystal structures, including HER2Fc-pertuzumab and HER2Fc-trastuzumab, have been subjected to CDI and DSSO crosslinking. Crosslinks established by CDI and DSSO between HER2Fc and pertuzumab accurately revealed their interaction interface. CDI crosslinking contributes more than DSSO because of its short spacer arm and high reactivity towards hydroxyl groups, demonstrating its capacity in protein interaction analysis. The correct binding domain cannot be revealed solely based on DSSO in the HER2Fc-trastuzumab complex, because domain proximity revealed by this 7-atom spacer linker cannot be directly translated as binding interfaces. As the first successful XL/MS application in early-stage therapeutic antibody discovery, we analyzed the molecular binding interface between HER2Fc and H-mab, an innovant drug candidate whose paratopes have not been studied yet. We predict that H-mab probably targets HER2 Domain I. The proposed XL/MS workflow can serve as an accurate, fast, and low-cost method to study the interaction between antibodies and large multi-domain antigens. SignificanceThis article described a fast, low-consumption approach based on chemical crosslinking mass spectrometry (XL/MS) using two linkers for binding domain determination in multidomain antigen-antibody complexes. Our results highlighted the higher importance of zero-length crosslinks established by CDI than 7-atom DSSO crosslinks, as residue proximity revealed by zero-length crosslinks is closely related to epitope-paratope interaction surfaces. Furthermore, the higher reactivity of CDI towards hydroxyl groups broadens the ranges of possible crosslinks, despite the necessity of delicate operation in CDI crosslinking. We suggest that all established CDI and DSSO crosslinks should be comprehensively considered for correct binding domain analysis because predictions solely based on DSSO might be ambiguous. We have determined the binding interface in the HER2-H-mab using CDI and DSSO, which is the first successful application of XL/MS in real-world early-stage biopharmaceutical development.

A Glycosidic-Bond-Based Mass-Spectrometry-Cleavable Cross-linker Enables In Vivo Cross-linking for Protein Complex Analysis.

Chemical cross-linking mass spectrometry (CXMS) has emerged as a powerful technology to analyze protein complexes. However, the progress of in vivo CXMS studies has been limited by cross-linking biocompatibility and data analysis. Herein, a glycosidic bond-based MS-cleavable cross-linker of trehalose disuccinimidyl ester (TDS) was designed and synthesized, which was fragmented in MS under CID/HCD to simplify the cross-linked peptides into conventional single peptides via selective cleavage between glycosidic and peptide bonds under individual MS collision energy. Consequently, the cross-linking identification accuracy and throughput were significantly enhanced, and the popular MS mode of stepped HCD was allowed. In addition, TDS showed proper cell-penetrating properties while being highly water-soluble, making it non-DMSO dependent during solubilization. Collectively, TDS provides a promising toolkit for CXMS characterization of living systems with high biocompatibility and accuracy.

Real-Time Library Search Increases Cross-Link Identification Depth across All Levels of Sample Complexity.

Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker-modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

Improved Analysis of Cross-Linking Mass Spectrometry Data with Kojak 2.0, Advanced by Integration into the Trans-Proteomic Pipeline.

Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of cross-links improve the sensitivity and accuracy of correct cross-link identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multiplatform.

Collagen Cross-Linking Lignin Improves the Bonding Performance of Etch-and-Rinse Adhesives to Dentin.

To evaluate the biomodification ability of lignin used as pre-treatment in human dentin before the application of an etch-and-rinse adhesive. Experimental hydroethanolic solutions with different cross-linking agents were used: 6.5% proanthocyanidins (PAC, from grape-seed extract); 2% cardanol (CARD, from cashew-nut shell liquid); lignin (LIG, from eucalyptus) at 1, 2 or 4% concentrations. The negative control (NC) was ethanol 50 v%. Extracted molars were prepared, and dentin microtensile bond strength (μTBS) was evaluated after 24 h water storage or 10,000 thermocycling aging. Further specimens were processed for SEM nanoleakage, micropermeability confocal microscopy evaluation and in situ degree of conversion (DC) through micro-Raman spectroscopy. Demineralized dentin sticks were submitted to a three-point bending test to evaluate the elastic modulus (E) before and after 1 min biomodification using the tested solutions. Moreover, it was also evaluated the mass changes and hydroxyproline (HYP) release after 4-weeks of water storage. Vibrational collagen crosslinking identification was evaluated through micro-Raman spectroscopy. The results were analyzed by analysis of variance (ANOVA) and Tukey’s test (α = 0.05). A significant reduction in μTBS was observed in groups NC (p < 0.001) and CARD (p = 0.026). LIG-4% showed no significant reduction in μTBS after aging (p = 0.022). Nanoleakage micrographs showed hybrid layer protection with all agents, but reduced micropermeability was attained only with lignin. Polymerization was negatively affected in the presence of all tested cross-linking agents, except LIG-1%. Lignin and cardanol increased the dentin E values, but only lignin reduced the mass loss in dentin specimens. Effective collagen crosslinking (1117 cm−1 and 1235 cm−1) was detected for all agents. HYP release was significantly lower with LIG-1% than NC (p < 0.001). Lignin was able to perform collagen cross-linking and prevent the degradation of unprotected dentin collagen, thereby improving the bonding performance of the composite restorations performed in this study.

Optimized TMT-Based Quantitative Cross-Linking Mass Spectrometry Strategy for Large-Scale Interactomic Studies.

Cross-linking mass spectrometry (XL-MS) is a powerful method for the investigation of protein-protein interactions (PPI) from highly complex samples. XL-MS combined with tandem mass tag (TMT) labeling holds the promise of large-scale PPI quantification. However, a robust and efficient TMT-based XL-MS quantification method has not yet been established due to the lack of a benchmarking dataset and thorough evaluation of various MS parameters. To tackle these limitations, we generate a two-interactome dataset by spiking in TMT-labeled cross-linked Escherichia coli lysate into TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme. Using this benchmarking dataset, we assess the efficacy of cross-link identification and accuracy of cross-link quantification using different MS acquisition strategies. For identification, we compare various MS2- and MS3-based XL-MS methods, and optimize stepped higher energy collisional dissociation (HCD) energies for TMT-labeled cross-links. We observed a need for notably higher fragmentation energies compared to unlabeled cross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3-, and synchronous precursor selection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vast number of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexed quantitative PPI characterization from complex biological systems.

Two-Dimensional Fractionation Method for Proteome-Wide Cross-Linking Mass Spectrometry Analysis.

Cross-linking mass spectrometry (XL-MS) is an emergent technology for studying protein-protein interactions (PPIs) and elucidating architectures of protein complexes. The development of various MS-cleavable cross-linkers has facilitated the identification of cross-linked peptides, enabling XL-MS studies at the systems level. However, the scope and depth of cellular networks revealed by current XL-MS technologies remain limited. Due to the inherently broad dynamic range and complexity of proteomes, interference from highly abundant proteins impedes the identification of low-abundance cross-linked peptides in complex samples. Thus, peptide enrichment prior to MS analysis is necessary to enhance cross-link identification for proteome-wide studies. Although chromatographic techniques including size exclusion (SEC) and strong cation exchange (SCX) have been successful in isolating cross-linked peptides, new fractionation methods are still needed to further improve the depth of PPI mapping. Here, we present a two-dimensional (2D) separation strategy by integrating peptide SEC with tip-based high pH reverse-phase (HpHt) fractionation to expand the coverage of proteome-wide XL-MS analyses. Combined with the MS-cleavable cross-linker DSSO, we have successfully mapped in vitro PPIs from HEK293 cell lysates with improved identification of cross-linked peptides compared to existing approaches. The method developed here is effective and can be generalized for cross-linking studies of complex samples.

Qualitative LC-MS/MS Identification, Formation and Stability of Adducts and Crosslinks Arising from the Reactions of Glutathione with the Model Enal Systems

Glutathione (GSH), due to the ability to capture the reactive electrophiles of exoand endogenous origins, is expected to prevent cross-linking induced by these compounds. However, it may instead become cross-linked itself. We subjected glutathione to reactions with model α,β-unsaturated carbonyl systems resulting from the interactions of adenosine with bifunctional aldehyde products of lipid peroxidation, and identified a range of adducts and cross-linked products. We found that the S-conjugated adducts, initially formed in the typical GSH Michael addition to α,β-unsaturated carbonyl system, unexpectedly undergo gradual degradation giving rise to the final N-conjugated products, in which formation of peptide amino group is involved instead of sulfhydryl functionality. This finding shows that the role of the GSH amino group in the non-enzymatic detoxification is underestimated, and that reactions between cellular α,β-unsaturated carbonyl compounds and GSH may be more complex than are presently perceived.

Facilitating In Situ Cross-Linking and Mass Spectrometry by Antibody-Based Protein Enrichment.

Cross-linking of living cells followed by mass spectrometry identification of cross-linked peptides (in situ CLMS) is an emerging technology to study protein structures in their native environment. One of the inherent difficulties of this technology is the high complexity of the samples following cell lysis. Currently, this difficulty largely limits the identification of cross-links to the more abundant proteins in the cell. Here, we describe a targeted approach in which an antibody is used to purify a specific protein-of-interest out of the cell lysate. Mass spectrometry analysis of the protein material that binds to the antibody can then identify considerably more cross-links on the target protein. By using an antibody against the CCT chaperonin, we identified over 200 cross-links that provide in situ evidence for the subunit arrangement of the CCT particle and its interactions with prefoldin. Similar targeting with an antibody against tubulin provided in situ evidence for the structure of the microtubule. Finally, the approach was also successful in identifying cross-links within a protein that expresses at a low level. These results demonstrate the general utility of antibody-based sample simplification for in situ CLMS and greatly expand the scope of protein systems that are amenable to in situ structural studies.

Characterizing protein conformers by cross-linking mass spectrometry and pattern recognition.

Chemical cross-linking coupled to mass spectrometry (XLMS) emerged as a powerful technique for studying protein structures and large-scale protein-protein interactions. Nonetheless, XLMS lacks software tailored toward dealing with multiple conformers; this scenario can lead to high-quality identifications that are mutually exclusive. This limitation hampers the applicability of XLMS in structural experiments of dynamic protein systems, where less abundant conformers of the target protein are expected in the sample. We present QUIN-XL, a software that uses unsupervised clustering to group cross-link identifications by their quantitative profile across multiple samples. QUIN-XL highlights regions of the protein or system presenting changes in its conformation when comparing different biological conditions. We demonstrate our software's usefulness by revisiting the HSP90 protein, comparing three of its different conformers. QUIN-XL's clusters correlate directly to known protein 3D structures of the conformers and therefore validates our software. QUIN-XL and a user tutorial are freely available at http://patternlabforproteomics.org/quinxl for academic users. Supplementary data are available at Bioinformatics online.

Identification of Native Cross-Links in Bacillus subtilis Spore Coat Proteins

The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis of the proteinaceous component of the spore coat, we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods. Young (day 1) and matured (day 5) Bacillus subtilis spores of wild-type and transglutaminase mutant strains were digested with formic acid and trypsin, and cross-linked peptides were enriched using strong cation exchange chromatography. The enriched cross-linked peptide fractions were subjected to Fourier-transform ion cyclotron resonance tandem mass spectrometry, and the high-quality fragmentation data obtained were analyzed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. This analysis identified specific disulfide bonds between coat proteins CotE–CotE and CotJA–CotJC, obtained evidence of disulfide bonds in the spore crust proteins CotX, CotY, and CotZ, and identified dityrosine and ε-(γ)-glutamyl-lysine cross-linked coat proteins. The findings in this Letter are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence of the cross-linked building blocks of the highly ordered and resistant structure called the spore coat.

Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass spectrometry.

Cross‐linking mass spectrometry has developed into an important method to study protein structures and interactions. The in‐solution cross‐linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross‐links obtained from co‐occurring protein oligomers, complexes, or conformers. Here we developed a cross‐linking workflow combining blue native PAGE with in‐gel cross‐linking mass spectrometry (IGX‐MS). This workflow circumvents steps, such as buffer exchange and cross‐linker concentration optimization. Additionally, IGX‐MS enables the parallel analysis of co‐occurring protein complexes using only small amounts of sample. Another benefit of IGX‐MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over‐length cross‐links compared to in‐solution cross‐linking. We next used IGX‐MS to investigate the complement components C5, C6, and their hetero‐dimeric C5b6 complex. The obtained cross‐links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX‐MS can provide new insights into the initial stages of the terminal complement pathway.

Expanding the Depth and Sensitivity of Cross-Link Identification by Differential Ion Mobility Using High-Field Asymmetric Waveform Ion Mobility Spectrometry.

In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the high complex sample compared to the analyses without FAIMS. Furthermore, we show that, for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with a comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein–protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies.

Use of Multiple Ion Fragmentation Methods to Identify Protein Cross-Links and Facilitate Comparison of Data Interpretation Algorithms.

Multiple ion fragmentation methods involving collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) with regular and very high energy settings, and electron-transfer dissociation with supplementary HCD (EThcD) are implemented to improve the confidence of cross-link identifications. Three different S. cerevisiae proteasome samples cross-linked by diethyl suberthioimidate (DEST) or bis(sulfosuccinimidyl)suberate (BS3) are analyzed. Two approaches are introduced to combine interpretations from the above four methods. Working with cleavable cross-linkers such as DEST, the first approach searches for cross-link diagnostic ions and consistency among the best interpretations derived from all four MS2 spectra associated with each precursor ion. Better agreement leads to a more definitive identification. Compatible with both cleavable and noncleavable cross-linkers such as BS3, the second approach multiplies scoring metrics from a number of fragmentation experiments to derive an overall best match. This significantly increases the scoring gap between the target and decoy matches. The validity of cross-links fragmented by HCD alone and identified by Kojak, MeroX, pLink, and Xi was evaluated using multiple fragmentation data. Possible ways to improve the identification credibility are discussed. Data are available via ProteomeXchange with identifier PXD018310.

Identifying transglutaminase reaction products via mass spectrometry as exemplified by the MUC2 mucin - Pitfalls and traps

In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.

MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a "MS2-centric" approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics ["fraction of mis-identifications" (FMI) and "fraction of interprotein cross-links from known interactions" (FKI)]. We then address this problem, by designing a novel "MS3-centric" approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.

The molecular architecture of photoreceptor phosphodiesterase 6 (PDE6) with activated G protein elucidates the mechanism of visual excitation

Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαβ) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gtα) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαβ in its nonactivated state. Following reconstitution of PDE6 and activated Gtα with liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα-binding sites per holoenzyme. Each Gtα interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.

The Isotope-Labeled, MS-Cleavable Cross-Linker Disuccinimidyl Dibutyric Urea for Improved Cross-Linking/Mass Spectrometry Studies.

Previous studies have shown the benefits of the amine-reactive, CID-MS/MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) for structural proteomics studies via cross-linking/MS (XL-MS). To further facilitate the automation of XL-MS experiments, we synthesized a deuterated (D12) version of the DSBU cross-linker combining the advantages of MS-cleavable linkers and isotope labeling. The rationale of conducting XL-MS with a mixture of unlabeled and stable isotope-labeled DSBU is to obtain characteristic mass differences at the MS level indicating cross-linked species. These cross-linked species can then be selected for fragmentation by collisional activation. At the MS/MS level, the characteristic 26-u doublets arising from cleavage of the central urea group in DSBU confirm the amino acid sequences of cross-linked peptides as well as the exact cross-linking sites. D12-labeled DSBU was tested on three systems with increasing complexity: (i) bovine serum albumin as purified protein, (ii) Escherichia coli ribosome as large, multimeric protein assembly, and (iii) Drosophila embryo extract as complete proteome. We demonstrate the benefits arising from the use of isotope-labeled DSBU for an automated assignment of cross-linked products. Combining isotope labeling and MS cleavability in one cross-linker resulted in higher cross-link identification numbers especially for highly complex protein mixtures.