The beginning of a beautiful friendship: cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes

After more than a decade of method development, cross-linking in combination with mass spectrometry and bioinformatics is finally coming of age. This technology now provides improved opportunities for modelling by mapping structural details of functional complexes in solution. The structure of proteins or protein complexes is ascertained by identifying amino acid pairs that are positioned in close proximity to each other. The validity of this technique has recently been benchmarked for large multi-protein complexes, by comparing cross-link data with that from a crystal structure of RNA polymerase II. Here, the specific nature of this cross-linking data will be discussed to assess the technical challenges and opportunities for model building. We believe that once remaining technological challenges of cross-linking/mass spectrometry have been addressed and cross-linking/mass spectrometry data has been incorporated into modelling algorithms it will quickly become an indispensable companion of protein and protein complex modelling and a corner-stone of integrated structural biology.     

Mapping spatial proximities of sulfhydryl groups in proteins using a fluorogenic cross-linker and mass spectrometry

Chemical cross-linking of proteins in combination with mass spectrometric analysis of the reaction products has gained renewed interest as a method of obtaining distance constraints within a protein and determining a low-resolution three-dimensional structure. We present a method for identifying spatially close sulfhydryl groups in proteins employing chemical cross-linking with the fluorogenic, homobifunctional cross-linker dibromobimane, which cross-links thiol pairs within approximately 3-6A. The applicability of our strategy was demonstrated by cross-linking the sulfhydryl groups of Cys-18 and Cys-78 in gamma-crystallin F, which are within a distance of 3.57A according to the X-ray structure. Intramolecularly cross-linked gamma-crystallin was first separated from reaction side products by reversed-phase chromatography on a C-4 column. Subsequently, the fraction containing the reacted protein was enzymatically digested with trypsin, and the resulting peptide mixture was separated by a second reversed-phase chromatographic step on a C-18 column, in which the cross-linked peptides were tracked by their fluorescence. The cross-linking product between Cys-18 and Cys-78 in gamma-crystallin F was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This strategy presents a rapid method for mapping sulfhydryl groups separated by a distance of approximately 3-6A within a protein.     

The advancement of chemical cross-linking and mass spectrometry for structural proteomics: from single proteins to protein interaction networks

During the last 15 years, chemical cross-linking combined with mass spectrometry (MS) and computational modeling has advanced from investigating 3D-structures of isolated proteins to deciphering protein interaction networks. In this article, the author discusses the advent, the development and the current status of the chemical cross-linking/MS strategy in the context of recent technological developments. A direct way to probe in vivo protein–protein interactions is by site-specific incorporation of genetically encoded photo-reactive amino acids or by non-directed incorporation of photo-reactive amino acids. As the chemical cross-linking/MS approach allows the capture of transient and weak interactions, it has the potential to become a routine technique for unraveling protein interaction networks in their natural cellular environment.     

Xlink-identifier: an automated data analysis platform for confident identifications of chemically cross-linked peptides using tandem mass spectrometry

Chemical cross-linking combined with mass spectrometry provides a powerful method for identifying protein-protein interactions and probing the structure of protein complexes. A number of strategies have been reported that take advantage of the high sensitivity and high resolution of modern mass spectrometers. Approaches typically include synthesis of novel cross-linking compounds, and/or isotopic labeling of the cross-linking reagent and/or protein, and label-free methods. We report Xlink-Identifier, a comprehensive data analysis platform that has been developed to support label-free analyses. It can identify interpeptide, intrapeptide, and deadend cross-links as well as underivatized peptides. The software streamlines data preprocessing, peptide scoring, and visualization and provides an overall data analysis strategy for studying protein-protein interactions and protein structure using mass spectrometry. The software has been evaluated using a custom synthesized cross-linking reagent that features an enrichment tag. Xlink-Identifier offers the potential to perform large-scale identifications of protein-protein interactions using tandem mass spectrometry.     

Identification of retrovirus matrix proteins by lipid-protein cross-linking.

Dimethyl suberimidate and its analogs are symmetrical bifunctional reagents that form amidine linkages with primary amino groups. These reagents have been used previously to study nearest neighbor relationships of proteins in viruses and in complex structures such as ribosomes. Dimethyl suberimidate also reacts with phosphatidylethanolamine, which can be radioactively labeled specifically with [¹⁴C]ethanolamine. When enveloped viruses containing radioactive phosphatidylethanolamine are exposed to the diimido ester, a fraction of the radioactivity becomes linked to viral structural proteins. Upon separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the lipid-protein complexes can be visualized on fluorograms of the gels. The cross-linking of lipids to proteins is specific, since it requires the viral structure to be intact, and since only certain proteins become chemically linked to phosphatidylethanolamine even though all the proteins react with dimethyl suberimidate. In vesicular stomatitis virus, the structure of which has been well characterized, only the glycoprotein and the matrix protein become linked to lipid. This is consistent with their known locations protruding outwards and inwards from the virus membrane, respectively. Thus we infer that the cross-linking technique can be used to identify proteins in close proximity to the lipid bilayer. In the avian leukemia and sarcoma viruses the protein designated p 19, and in the murine leukemia viruses p 15 become linked to radioactive lipid. Since avian p19 and murine p15 are internal structural proteins, we infer that they are equivalent to the matrix protein defined for other other enveloped viruses.     

Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics

Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.The concept of protein cross-linking as a (bio)chemical tool to infer structural information about protein conformations and protein-protein interactions in combination with mass spectrometry was introduced at the end of the 1990s (1). In a seminal paper, Young et al. (1) used chemical cross-linking of lysine residues in bovine basic fibroblast growth factor FGF-2 (heparin-binding growth factor 2) to provide distance constraints for the computational derivation of the fold of this small (17-kDa) protein. FGF-2 was cross-linked with bis(sulfosuccinimidyl) suberate, purified by size exclusion chromatography, and digested with trypsin. Cross-linked peptides were separated by HPLC and analyzed on line by ESI-TOF and off line by MALDI-TOF mass spectrometry. Putative cross-links were then assigned based on their precursor masses, and some of them were verified by MALDI postsource decay. The authors could identify 15 cross-links that did not bridge directly adjacent lysines and therefore provided information on the three-dimensional structure of the protein. These data were used to assign FGF-2 to the β-trefoil family by excluding calculated models that did not fit the distance constraints.In the last decade, the application of protein cross-linking has expanded, first and foremost driven by developments in mass spectrometry as the method of choice for the high throughput identification of proteins and their modifications. Reviews by Back et al. (2), Sinz (3), and most recently Lee (4) give an overview on the evolution of the field. However, despite the progress that has undoubtedly been made, cross-linking is still considered a “niche” technique that has not (yet) lived up to its promises. High throughput generation of data supporting protein fold prediction and the determination of protein-protein interactions have not been realized routinely. There may be several reasons for that such as the necessity of access to high end mass spectrometers, the requirement of specialized reagents, and the need for tailored software. However, recent years have seen an increased interest in this technique, which is reflected in the literature and by the emergence of new reagents and software tools.Here, we present an overview of recent developments in methodology, instrumentation, and bioinformatics related to chemical cross-linking of proteins and the analysis of cross-linked peptides by mass spectrometry. Other cross-linking areas such as protein-DNA cross-linking, photoinduced cross-linking, or the characterization of disulfide bonds will not be covered in detail in this paper. We critically discuss advantages and limitations of different concepts and look beyond the immediate outcome of cross-linking experiments (putative interactions and/or distance constraints) and examine the potential role of chemical cross-linking in the analysis of protein interaction networks and, more generally, for structural and systems biology.     

Purifying protein complexes for mass spectrometry: applications to protein translation

Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.     

"Zero-length" cross-linking in solid state as an approach for analysis of protein-protein interactions

We have developed a new approach for the analysis of interacting interfaces in protein complexes and protein quaternary structure based on cross-linking in the solid state. Protein complexes are freeze-dried under vacuum, and cross-links are introduced in the solid phase by dehydrating the protein in a nonaqueous solvent creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross-linked proteins are digested into peptides with trypsin in both H2(16)O and H(2)18O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross-linked peptides. We demonstrated specificity and reproducibility of our method by cross-linking homo-oligomeric protein complexes of glutathione-S-transferase (GST) from Schistosoma japonicum alone or in a mixture of many other proteins. Identified cross-links were predominantly of amide origin, but six esters and thioesters were also found. The cross-linked peptides were validated against the GST monomer and dimer X-ray structures and by experimental (MS/MS) analyses. Some of the identified cross-links matched interacting peptides in the native 3D structure of GST, indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze-drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru (II) Bpy(3)2+ catalyzed, oxidative "zero-length" cross-linking, confirming that it is feasible to use our strategy for analyzing the molecular interfaces of interacting proteins or peptides.     

Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein-protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer software. This method is specifically tailored toward the rapid analysis of low microgram amounts of proteins or multimeric protein complexes cross-linked with nonlabeled and deuterium-labeled bis-NHS ester cross-linking reagents (both commercially available and readily synthesized). Through labeling with [18O]water solvent and LC-MALDI analysis, the method further allows the possible distinction between Type 0 and Type 1 or Type 2 modified peptides (monolinks and looplinks or cross-links), although such a distinction is more readily made from analysis of tandem mass spectrometry data. When applied to the bacterial Colicin E7 DNAse/Im7 heterodimeric protein complex, 23 cross-links were identified including six intersubunit cross-links, all between residues that are close in space when examined in the context of the X-ray structure of the heterodimer. In addition, cross-links were successfully identified in five single subunit proteins, beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A, establishing the generality of the approach.     

Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins

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 cross-linked 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 (PrPβ). 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 cross-linked 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(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.     

Covalent blocking of fibril formation and aggregation of intracellular amyloidgenic proteins by transglutaminase-catalyzed intramolecular cross-linking

Two different types of physical bonding have been proposed to involve in the formation of neuronal inclusions of patients with neurodegenerative diseases such as Alzheimer's, Parkinson's, and polyglutamine diseases. One is the noncovalent bonding that stabilizes the amyloid-type fibrous aggregates, and the other is the covalent cross-linking catalyzed by tissue transglutaminase. The cross-linking is subdivided into the inter- and intramolecular cross-linking. Little attention has been paid to the pathological roles of the intramolecular cross-linking. To elucidate the possible interplay between the intramolecular cross-linking and the amyloid-type fibril formation, we performed an in vitro aggregation analysis of three intracellular amyloidgenic proteins (a domain of tau protein, alpha-synuclein, and truncated yeast prion Sup35) in the presence of tissue transglutaminase. The analysis was performed in low concentrations of the proteins using techniques including thioflavin T binding and mass spectrometry. The results demonstrated that the amyloid-type fibril formation was strongly inhibited by the transglutaminase-catalyzed intramolecular cross-linking, which blocked both the nucleation and the fiber extension steps of the amyloid formation. Far-UV CD spectroscopy indicated that the cross-linking slightly altered the backbone conformation of the proteins. It is likely that conformational restriction imposed by the intramolecular cross-links has impaired the ordered assembly of the amyloidgenic proteins. Nonamyloid type aggregation was also suppressed by the intramolecular cross-links. On the basis of the results, we proposed that tissue transglutaminase is a modulator for the protein aggregation and can act defensively against the fibril deposition in neurons.     

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.     

Chemical cross-linking and mass spectrometry for protein structural modeling

The growth of gene and protein sequence information is currently so rapid that three-dimensional structural information is lacking for the overwhelming majority of known proteins. In this review, efforts towards rapid and sensitive methods for protein structural characterization are described, complementing existing technologies. Based on chemical cross-linking and offering the analytical speed and sensitivity of mass spectrometry these methodologies are thought to contribute valuable tools towards future high throughput protein structure elucidation.     

Structure and dynamics of dark-state bovine rhodopsin revealed by chemical cross-linking and high-resolution mass spectrometry

Recent work using chemical cross-linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G-protein-coupled receptor bovine rhodopsin in its native membrane using lysine- and cysteine-targeted bifunctional cross-linking reagents. Cross-linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT-ICR mass spectrometry with automated data-reduction and assignment software. Tandem mass spectrometry was used to verify cross-link assignments and locate the exact sites of cross-link attachment. Cross-links were observed to form between 10 pairs of residues in dark-state rhodopsin. For each pair, cross-linkers with a range of linker lengths were tested to determine an experimental distance-of-closest-approach (DCA) between reactive side-chain atoms. In all, 28 cross-links were identified using seven different cross-linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross-link between C316 and K325 in the C-terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross-linking reactions. Biochemical and spectroscopic data from other studies have found that the C-terminal region is highly mobile in solution and not fully represented by X-ray crystallography data. Our results show that chemical cross-linking can provide reliable three-dimensional structural information and insight into local conformational dynamics in a membrane protein.     

Characterization of acrolein-induced protein cross-links

Lipid peroxidation products contribute to protein aggregation that occurs during oxidative stress in a number of degenerative disorders. Acrolein (ACR), a highly toxic lipid peroxidation aldehyde, is a strong cross-linking agent of cellular components such as proteins. To understand the mechanisms of oxidative stress-induced protein aggregation, this study characterized the ACR modification of chain B from bovine insulin by mass spectrometry. To identify the cross-linking sites, the ACR-treated peptide was digested with a protease and the resulting peptides were analysed by liquid chromatography-tandem mass spectrometry. Inter- and intra-molecular cross-linking adducts were identified between amino groups and the side chain of histidine in the peptide. These results indicated that the ACR-induced cross-links were accompanied by two reactions, namely Michael addition and Schiff base formation. In conclusion, the use of mass spectrometric techniques provided chemical evidence for protein cross-linking with ACR.     

Identification and mapping of protein-protein interactions by a combination of cross-linking, cleavage, and proteomics

Protein-protein interactions are vital for almost all cellular functions, and many require the formation of multiprotein complexes. Identification of the macroscopic and microscopic protein interactions within these complexes is essential in understanding their mechanisms, both under physiologic as well as pathologic conditions. This review describes the current technology available to investigate interactions between proteins utilizing chemical cross-linking and site-directed cleavage reagents, outlining the necessary steps involved in identifying interacting proteins both in vitro and in vivo. Once interacting proteins are identified, more information about the architecture of the assemblies is necessary. Unique separation techniques coupled with cross-linking and mass spectrometry can now identify specific interaction sites and lead to the development of quaternary structural protein models. Furthermore, combination of these methods with proteomic approaches enables the identification and analysis of complex interactions in vivo. Finally, future directions in cross-linking methodologies are discussed.     

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.     

Characterization of acrolein-induced protein cross-links

Lipid peroxidation products contribute to protein aggregation that occurs during oxidative stress in a number of degenerative disorders. Acrolein (ACR), a highly toxic lipid peroxidation aldehyde, is a strong cross-linking agent of cellular components such as proteins. To understand the mechanisms of oxidative stress-induced protein aggregation, this study characterized the ACR modification of chain B from bovine insulin by mass spectrometry. To identify the cross-linking sites, the ACR-treated peptide was digested with a protease and the resulting peptides were analysed by liquid chromatography-tandem mass spectrometry. Inter- and intra-molecular cross-linking adducts were identified between amino groups and the side chain of histidine in the peptide. These results indicated that the ACR-induced cross-links were accompanied by two reactions, namely Michael addition and Schiff base formation. In conclusion, the use of mass spectrometric techniques provided chemical evidence for protein cross-linking with ACR.     

Structural complementarity of distance constraints obtained from chemical cross-linking and amino acid coevolution

The analysis of amino acid coevolution has emerged as a practical method for protein structural modeling by providing structural contact information from alignments of amino acid sequences. In parallel, chemical cross-linking/mass spectrometry (XLMS) has gained attention as a universally applicable method for obtaining low-resolution distance constraints to model the quaternary arrangements of proteins, and more recently even protein tertiary structures. Here, we show that the structural information obtained by XLMS and coevolutionary analysis are effectively complementary: the distance constraints obtained by each method are almost exclusively associated with non-coincident pairs of residues, and modeling results obtained by the combination of both sets are improved relative to considering the same total number of constraints of a single type. The structural rationale behind the complementarity of the distance constraints is discussed and illustrated for a representative set of proteins with different sizes and folds.