Patent Grant
10697977
The present application is being filed along with a Sequence Listing as an ASCII text file via EFS-Web. The Sequence Listing is provided as a file entitled UCI012003ASEQLIST.txt, created and last saved on Jun. 2, 2017, which is 58,182 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety in accordance with 35 U.S.C. § 1.52(e).
The present disclosure relates generally to mass spectrometry-cleavable cross-linkers and methods of using mass spectrometry-cleavable cross-linkers for probing proteins and protein complexes.
The majority of proteins exert their functions in the form of protein complexes. These macromolecular assemblies and their protein-protein interactions play critical roles in regulating integral biological processes. As a result, perturbations of endogenous protein-protein interactions can result in deleterious effects on cellular activities.
Structural analyses of these complexes by traditional biophysical structural techniques such as x-ray crystallography and nuclear magnetic resonance (NMR) are frequently utilized to elucidate their topologies. Unfortunately, many large and heterogeneous complexes are refractory to such methods, ushering the development of new hybrid structural strategies.
In some embodiments, an MS-cleavable cross-linker is provided. In some embodiments, the MS-cleavable cross-linker comprises at least one hydrazide reactive group, at least one sulfoxide group, and at least one collision-induced dissociation (CID) cleavable bond, wherein the MS-cleavable cross-linker is configured for mapping intra-protein interactions in a protein, or inter-protein interactions in a protein complex, or combinations thereof.
In some embodiments of the MS-cleavable cross-linker, the at least one hydrazide reactive group is configured to react with an activated acidic side chain in the protein or protein complex. In some embodiments of the MS-cleavable cross-linker, the at least one CID cleavable bond is a C—S bond adjacent to the at least one sulfoxide group. In some embodiments of the MS-cleavable cross-linker, the MS-cleavable cross-linker is dihydrazide sulfoxide (DHSO), having the structure:
In some embodiments of the MS-cleavable cross-linker, the two hydrazide reactive groups are separated by a 12.5 Å long spacer arm comprising the two symmetrical CID cleavable C—S flanking the one sulfoxide group.
In some embodiments, a method for synthesis of an MS-cleavable cross-linker is provided. In some embodiments, the method for synthesis of an MS-cleavable cross-linker comprises the steps of (i) providing disuccinimidyl sulfoxide (DSSO) in dicholormethane, (ii) adding tert-butyl carbazate to derive a first solution, (iii) stirring the first solution of step (ii), (iv) adding trifluoroacetic acid to derive an second solution, (v) stirring the second solution of step (iv), (vi) removing solvent from the second solution of step (v) in vacuo to derive an oil, (vii) dissolving the oil from step (vi) in methanol and adding trimethylamine to obtain a mixture, (viii) stirring the mixture from step (vii) to obtain a precipitate, (ix) collecting the precipitate from step (viii), washing the precipitate in fresh methanol, and drying the precipitate in vacuo, thereby obtaining the MS-cleavable cross-linker.
In some embodiments of the method for synthesis, stirring the first solution of step (ii) is performed at room temperature. In some embodiments of the method for synthesis, stirring the first solution of step (ii) is performed for about 6 h to about 24 h. In some embodiments of the method for synthesis, stirring the second solution of step (iv) is performed at room temperature. In some embodiments of the method for synthesis, stirring the second solution of step (iv) is performed for about 36 h to about 144 h. In some embodiments of the method for synthesis, stirring the mixture is performed at room temperature. In some embodiments of the method for synthesis, stirring the mixture is performed for about 10 min to about 40 min. In some embodiments of the method for synthesis, collecting the precipitate from step (viii), washing the precipitate in fresh methanol is performed about 1 to about 10 times. In some embodiments of the method for synthesis, the MS-cleavable cross-linker is DHSO, having the structure:
In some embodiments, a method for mapping intra-protein interactions in a protein, inter-protein interactions in a protein complex, or combinations thereof is provided. In some embodiments, the method for mapping intra-protein interactions comprises providing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as an activating agent, activating at least one acidic residue by DMTMM in the protein or protein complex, providing an MS-cleavable cross-linker, wherein the MS-cleavable cross-linker comprises at least one hydrazide reactive group, at least one sulfoxide group, and at least one CID cleavable bond, cross-linking the MS-cleavable cross-linker to the at least one activated acidic residue in the protein or protein complex, digesting with an enzyme the protein or protein complex cross-linked to the MS-cleavable cross-linker, generating one or more peptide fragments of the protein or protein complex, wherein the one or more peptide fragments are chemically cross-linked to the MS-cleavable cross-linker, and identifying the one or more peptide fragments using tandem mass spectrometry (MSn), thereby mapping intra-protein interactions in the protein and/or inter-protein interactions in the protein complex.
In some embodiments of the method for mapping intra-protein interactions, the MS-cleavable cross-linker is DHSO, having the structure
In some embodiments, a method for cross-linking mass spectrometry (XL-MS) for identifying one or more cross-linked peptides is provided. In some embodiments, the method for XL-MS comprises providing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as an activating agent, activating at least one acidic residue by DMTMM in a protein or a protein complex, providing an MS-cleavable cross-linker, wherein the MS-cleavable cross-linker comprises at least one hydrazide reactive group, at least one sulfoxide group, and at least one CID cleavable bond, cross-linking the MS-cleavable cross-linker to the at least one activated acidic residue in the protein or protein complex, digesting with an enzyme the protein or protein complex cross-linked to the MS-cleavable cross-linker, generating one or more peptide fragments of the protein or protein complex, wherein the one or more peptide fragments are chemically cross-linked to the MS-cleavable cross-linker, performing a liquid chromatography-tandem mass spectrometry (LC-MSn) analysis on the one or more cross-linked peptides, wherein the LC-MS1 analysis comprises detecting the one or more cross-linked peptides by MS1 analysis, selecting the one or more cross-linked peptides detected by MS1 for MS2 analysis, selectively fragmenting the at least one CID cleavable bond and separating the one or more cross-linked peptides during MS2 analysis, sequencing the one or more cross-linked peptides separated during MS2 analysis by MS3 analysis, and integrating data obtained during MS1, MS2 and MS3 analyses to identify the one or more cross-linked peptides.
In some embodiment of the method for XL-MS, the MS-cleavable cross-linker is DHSO, having the structure
The majority of proteins exert their functions in the form of protein complexes. These macromolecular assemblies and their protein-protein interactions play critical roles in regulating integral biological processes. As a result, perturbations of endogenous protein-protein interactions can result in deleterious effects on cellular activities. Structural analyses of these complexes by traditional biophysical structural techniques such as x-ray crystallography and nuclear magnetic resonance (NMR) are frequently utilized to elucidate their topologies. Unfortunately, many large and heterogeneous complexes are refractory to such methods, ushering the development of new hybrid structural strategies.
Chemical cross-linking coupled with mass spectrometry (XL-MS) has emerged as a powerful and popular approach for delineating the protein interactions within large multi-subunit protein complexes1,2. Moreover, cross-linking can capture temporal protein interactions by forming covalent bonds between proximal amino acid residues, effectively freezing transient interactions and providing information on the identities and spatial orientations of interacting proteins simultaneously. These linkages are then utilized as distance constraints to facilitate three-dimensional modeling of protein complexes by refining existing high-resolution protein structures or complementing lower resolution biophysical structural techniques (e.g. cryo-electron microscopy) in order to position individual protein subunits or interacting regions3-9.
Elucidating the structure of protein complexes is paramount for understanding their functions. Recent technological advancements in cross-linking mass spectrometry (XL-MS) have made it a powerful methodology for defining protein-protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. One of the major challenges in conventional XL-MS studies is unambiguous identification of cross-linked peptides, due to difficulty in interpreting convoluted tandem mass spectra resulting from the fragmentation of covalently linked peptides.
To this end, various types of cleavable cross-linkers have been developed to facilitate and simplify MS identification of cross-linked peptides, such as UV-photocleavable10, chemical-cleavable11, and MS-cleavable reagents12-18. Of these, MS-cleavable cross-linkers appear to be the most attractive option due to their capability to improve the identification of cross-linked peptides by separating inter-linked peptides during MS analysis.
To facilitate this process, the inventors have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed the inventors to establish a common robust XL-MS workflow (for example,
These MS-cleavable reagents contain symmetric MS-labile C—S bonds (adjacent to the sulfoxide group) that can be selectively and preferentially fragmented prior to peptide backbone cleavage during collision induced dissociation (CID)16-18. Such fragmentation is robust and predictable, occurring independently of cross-linking types, peptide charges, and sequences. Ultimately this unique feature enables simplified and unambiguous identification of cross-linked peptides by MSn analysis and conventional database searching tools16-18. In some embodiments, the inventors' sulfoxide-containing, MS-cleavable cross-linkers have been successfully applied to not only define protein-protein interactions and elucidate structures of protein complexes in vitro5,9,16,27 and in vivo18, but also quantify structural dynamics of protein complexes17,19.
In current XL-MS studies, amine-reactive reagents targeting lysine residues are the most widely used compounds for successful elucidation of protein structures. This is due to their effective and specific cross-linking chemistry, as well as frequent occurrence of lysine residues in protein sequences and at surface-exposed regions of protein structures. However, it remains challenging to characterize protein interaction interfaces with little to no lysine residues.
Therefore, there is a necessity for the development of additional cross-linking chemistries in order to increase the coverage of structural information obtainable from XL-MS experiments, particularly in systems where protein interacting regions are refractive to amine-specific cross-linking. Although several types of cross-linkers targeting other amino acids (e.g. sulfhydryl-reactive and non-specific photo-reactive) reagents are commercially available, their applications in studying protein-protein interactions thus far are very limited.
For instance, sulfhydryl-reactive cross-linking reagents carrying functional groups with specific chemistries targeting cysteine residues have not been widely adopted, most likely owing to the relatively low occurrence of cysteine residues and their participation in forming disulfide bonds in protein structures. In comparison, although photochemical cross-linking reagents can improve the coverage of protein interaction contacts by reacting with any amino acids non-specifically20, the resulting cross-linked products are often unpredictable, thus making their unambiguous MS identification even more difficult. In addition, such non-specific cross-linking has higher chance of introducing more non-specific interactions.
Therefore, a specific cross-linking chemistry targeting amino acid residues abundant at protein interaction sites would be ideal for complementing lysine targeting cross-linkers. While hydrophobic amino acid residues often constitute the cores, charged hydrophilic residues such as lysine, arginine, aspartic acid (Asp), and glutamic acid (Glu) often occupy surface-exposed regions of protein complexes, making them ideal targets for mapping protein interactions.
A recent study by Leitner et al. have demonstrated the feasibility of an acidic residue-specific cross-linking chemistry to study protein interactions using non-cleavable homobifunctional dihydrazide cross-linkers in conjunction with the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)22. This methodology is an improvement on the acidic residue cross-linking chemistry involving the coupling reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) that requires the cross-linking reaction to occur at a pH of 5.523.
In comparison, DMTMM coupling with dihydrazide cross-linkers is compatible with proteins at neutral pH (7.0-7.5) and therefore better suited for studying the structures of proteins and protein complexes under physiological conditions. However, this cross-linking strategy remains susceptible to the challenges associated with traditional cross-linking reagents in unambiguously identifying cross-linked peptides and their linkage sites. Due to the increased prevalence of Asp and Glu in protein sequences, the accurate and unambiguous identification of peptides containing non-cleavable dihydrazide cross-linked acidic residues would be intrinsically more complicated than the identification of lysine cross-linked peptides.
Due to the lack of proper reagents targeting other residues, it remains challenging to characterize protein interaction interfaces with little or no lysine residues. Similar to lysine residues, acidic residues are abundant and often present at protein contact regions.
To expand the coverage of interaction regions by XL-MS studies, in some embodiments herein, a new acidic residue reactive and sulfoxide containing MS-cleavable cross-linker, 3,3′-sulfinyldi(propanehydrazide) (DHSO) is provided. The development of DHSO cross-linker not only enlarges the range of the application of XL-MS approaches, but also further demonstrates the robustness and applicability of sulfoxide-based MS cleavability in conjunction with various reactive chemistries. In comparison to existing non-cleavable dihydrazides, DHSO contains robust MS-cleavable bonds that enable simplified and unambiguous identification of DHSO cross-linked peptides.
To simplify MS analysis and facilitate the identification of acidic residue cross-linked peptides, a sulfoxide-containing, MS-cleavable, acidic residue-specific homobifunctional cross-linking reagent, dihydrazide sulfoxide (DHSO, a.k.a. 3,3′-sulfinyldi(propanehydrazide)) has been developed as disclosed herein. This reagent adopts the same MS-labile sulfoxide chemistry as the inventors' previously developed amine-reactive, MS-cleavable cross-linkers (i.e. DSSO, DMDSSO and Azide-A-DSBSO), thus enabling robust and unambiguous identification of cross-linked peptides via the same XL-MSn workflow16-18. DHSO represents a novel class and the first generation of acidic residue-targeting cross-linking reagents with MS-cleavability.
According to a recent SwissProt database release21; aspartic and glutamic acids comprise roughly 12.2% of all amino acid residues, compared to the 5.8% of lysines. Therefore, acidic residues (i.e. aspartic and glutamic acids) represent high potential targets for cross-linking studies due to their abundance and prevalence at interaction interfaces. In some embodiments, the proteins and/or protein complexes targeted by DHSO comprise about 1% to about 12.5% acidic amino acid residues. In some embodiments, the proteins and/or protein complexes targeted by DHSO comprise about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 22.5 or 25% acidic amino acid residues, or value within a range defined by any two of the aforementioned values.
In some embodiments, DHSO expands the coverage of protein interaction regions, disclosed herein is the development of a new acidic acid residue targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). In some embodiments, a novel acidic residue reactive and sulfoxide-containing MS-cleavable homobifunctional cross-linker for probing protein-protein interactions is provided28.
In some embodiments, DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as those amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. In some embodiments, proteins are cross-linked by DHSO (3,3′-sulfinyldi(propanehydrazide)) in the presence of coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), forming covalent linkages between proximal and interacting proteins through aspartic acid and/or glutamic acid linkages. The resulting cross-linked peptides were analyzed by multistage tandem mass spectrometry (MSn). In some embodiments, other acidic amino acids besides Asp and Glu are included within the scope of this disclosure.
Additionally, in some embodiments, DHSO can provide complimentary data to amine reactive reagents. The present disclosure expands the range of the application of XL-MS approaches, but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various reactive chemistries.
In some embodiments, an integrated Xl-MS platform to determine composition-dependent conformational changes of proteins and/or protein complexes is provided. In some embodiments, it is expected that DHSO-based XL-MS strategies will become an invaluable tool in providing a complementary subset of cross-linking data towards a comprehensive structural elucidation of protein complexes by XL-MS.
Design and Synthesis of a Novel Acidic Residue-Targeting Sulfoxide-Containing MS-Cleavable Cross-Linker
In order to facilitate facile and accurate identification of acidic residue cross-linked peptides, it was aimed to develop a novel MS-cleavable cross-linking reagent specific to Asp and Glu residues. In some embodiments, this required the incorporation of a functional group with robust MS-inducible cleavage sites located in the spacer region of the cross-linker. Previously, we successfully developed a novel class of amine-reactive, sulfoxide-containing MS-cleavable cross-linkers, i.e., DSSO16, DMDSSO17 and Azide-A-DSBSO18 (
It will be understood by one of ordinary skill in the art that the parameters and conditions provided herein are exemplary and are not limiting in any way. The parameters and conditions of synthesis can be adjusted by one of ordinary skill in the art based on need and the scale of synthesis desired. In some embodiments, disuccinimidyl sulfoxide (DSSO) is synthesized as previously published16. The 2-step synthesis scheme for DHSO from DSSO is depicted in
As shown below and in
In comparison to existing cross-linkers for XL-MS studies16-18,22, DHSO carries a linker length well suited for defining interaction interfaces between and within protein complexes.
In some embodiments, DHSO was synthesized and characterized with synthetic peptides and simple model proteins to confirm the features of its design. In some embodiments, the MS cleavability was validated by MSn analysis. In some embodiments, the DHSO based cross-linking mass spectrometry workflow provided herein has proven to be as effective as those used for inventors' previously developed sulfoxide-containing amine reactive MS-cleavable cross-linkers, i.e. DSSO, DMDSSO and Azide-A-DSBSO. In some embodiments, the DHSO-based cross-linking mass spectrometry workflow can complement lysine reactive cross-linkers for in vitro and in vivo cross-linking studies.
In some embodiments, DHSO represents the first MS-cleavable cross-linker targeting acidic residues. DHSO comprises robust MS-cleavable bonds, i.e. C—S bonds adjacent to the center sulfoxide group, which enables fast, effective, and accurate identification of DHSO cross-linked peptides. In some embodiments, based on analysis of the standard protein BSA, many more cross-linked peptides were identified with DHSO in comparison to existing non-cleavable dihydrazide reagents. In some embodiments, DHSO cross-linked peptides possess the same characteristics distinctive to peptides cross-linked by other sulfoxide-containing amine reactive cross-linkers previously developed by the inventors.
In some embodiments, the unique features of DHSO significantly facilitate cross-linking studies targeting acidic residues, which has been difficult in the past due to the large number of D/E present in protein sequences and complexity of their resulting cross-linked peptides for MS analysis. Comparison of DHSO and DSSO confirms the need of expanding the coverage of protein interactions using cross-linkers targeting different residues, especially when the distribution of specific amino acids is uneven. Thus, in some embodiments, development of DHSO crosslinking will aid in the goal of defining protein-protein interactions at the global scale and understanding the structural dynamics of protein complexes and their mechanistic functions in cells. In some embodiments, the DHSO-based workflow provided herein can be applied to cross-linking studies of simple and complex protein mixtures in vitro and in vivo.
CID Fragmentation Patterns of DHSO Cross-Linked Peptides
A previous study showed that the reaction of hydrazide cross-linkers with acidic residues first requires activation of the terminal carboxyl groups of Asp (D) and Glu (E) side chains or protein C-termimi22. The coupling reagent DMTMM was demonstrated to be effective in activating carboxylic acid groups to form a reactive intermediate that can be displaced by nucleophilic attack from hydrazides under physiological pH22 (
Since all of the MS-cleavable, homobifunctional NHS esters we previously developed display the same characteristic fragmentation patterns in MS2 due to the cleavage of either of the two symmetric CID-cleavable C—S bonds adjacent to the sulfoxide functional group16-18, it is expected that DHSO cross-linked peptides will behave similarly during MSn analysis even though their residue-targeting functional groups are different.
To elaborate this process,
Characterization of DHSO Cross-Linked Model Peptides by MSn Analysis
Despite the similarities in spacer arm structure to DSSO, it is necessary to verify whether DHSO cross-linked peptides indeed fragment the same way as described above during MSn analysis (
Characterization of DHSO Cross-Linked Model Proteins by MSn Analysis
To evaluate the capability of DHSO for protein cross-linking in vitro, equine myoglobin and bovine serum albumin (BSA) were used as model proteins. These two proteins contain above-average acidic residue content (16.3% and 13.6%, respectively), making them well suited for evaluating DHSO cross-linking. In addition, BSA was employed previously for acidic residue cross-linking by non-cleavable dihydrazides22. To identify DHSO cross-linked peptides in myoglobin and BSA, in-gel digestion of gel-separated DHSO cross-linked proteins or in solution digestion of DHSO cross-linked proteins followed by peptide SEC was performed as illustrated (
In addition to inter-linked peptides, intra-linked peptides were also observed as a result of DHSO cross-linking in the model proteins. For example, MS2 fragmentation of an intra-linked myoglobin peptide (
Moreover, MS2 fragmentation of a myoglobin dead-end modified peptide (m/z 604.30953+) resulted in the detection of a single pair of fragment ions αA/αT (m/z 559.303+/569.963+), consistent with the expected fragmentation pattern described in
In total, LC-MSn analysis of DHSO cross-linked myoglobin identified a total of 33 unique inter-linked peptides, representing 32 unique E|D-E|D linkages (Table 1).
Similarly, 62 unique DHSO inter-linked BSA peptides were identified, describing 69 unique E|D-E|D linkages (Table 2).
In some embodiments, the number of unique peptides obtained depends on several factors, including but not limited to, size, complexity, conformation, subunits, domains, etc., of the protein and/or protein complex. In some embodiments, the number of unique peptides range from about 5 to about 500. In some embodiments, the number of unique peptides range from about 10 to about 1000. In some embodiments, the number of unique peptides range from about 50 to about 5000. In some embodiments, the number of unique peptides range from about 500 to about 50,000. Collectively, the results presented thus far indicate that DHSO can effectively cross-link acidic residue containing peptides and proteins at neutral pH in the presence of DMTMM as the activating agent. More importantly, in some embodiments, the results demonstrate that DHSO cross-linked peptides indeed exhibit the same characteristic MS2 fragmentation patterns as expected to allow their facile and accurate identification.
DHSO Cross-Linking Maps of Myoglobin and BSA
In order to assess the efficacy and sequence coverage of DHSO cross-linking on model proteins, cross-linking maps of myoglobin and BSA based on the identified DHSO inter-linked peptides were generated. The secondary structures of equine myoglobin comprise of eight α-helices and one short 310 helix (PDB: 1DWR) (
Similarly, a DHSO cross-link map of BSA was generated based on the 69 unique E|D-E|D linkages (
In some embodiments, the Cα-Cα distance ranges from about 5 Å to about 50 Å. In some embodiments, the Cα-Cα distance ranges from about 1 Å to about 100 Å. In some embodiments, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 0-5 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 5-10 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 10-15 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 15-20 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 20-25 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 25-30 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 30-35 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 35-40 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 40-45 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 45-50 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 50-55 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 55-60 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 60-65 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 65-70 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 70-75 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 75-80 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 80-85 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 85-90 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 90-95 Å, the number of cross-links range from about 1 to about 100. In some embodiments, for a Cα-Cα distance range of about 95-100 Å, the number of cross-links range from about 1 to about 100. In some embodiments, the number of cross-links range from about 100 to about 10,000 for one or more of the Cα-Cα distance ranges disclosed herein. In some embodiments, about 5% to about 45% of intra-links are within any of the ranges disclosed herein. In some embodiments, about 25% to about 65% of intra-links are within any of the ranges disclosed herein. In some embodiments, about 45% to about 99% of intra-links are within any of the ranges disclosed herein. In some embodiments, about 5% to about 45% of inter-links are within any of the ranges disclosed herein. In some embodiments, about 25% to about 65% of inter-links are within any of the ranges disclosed herein. In some embodiments, about 45% to about 99% of inter-links are within any of the ranges disclosed herein. In some embodiments, about 5% to about 45% of intra- and inter-links are within any of the ranges disclosed herein. In some embodiments, about 25% to about 65% of intra- and inter-links are within any of the ranges disclosed herein. In some embodiments, about 45% to about 99% of intra- and inter-links are within any of the ranges disclosed herein.
As shown in
Comparison of MS-Cleavable and Non-Cleavable Acidic Residue Cross-Linking
Previously, two non-cleavable acidic residue cross-linkers, i.e., adipic acid dihydrazide (ADH) and pimetic acid dihydrazide (PDH), were used for probing the structure of bovine serum albumin22, which identified 27 and 35 unique acidic residue linkages, respectively22. A comparison of the linkage maps generated for DHSO, ADH, and PDH cross-linking of BSA (
Comparison of DHSO and DSSO Cross-Linking
To assess the complementarity between acidic residue and primary amine cross-linking data, the similarities and differences between DHSO and DSSO cross-linking of selected model proteins were examined. To this end, LC-MSn analyses of DSSO cross-linked myoglobin and BSA respectively were performed. As summarized in Table 3 and Table 4, 19 unique DSSO inter-linked myoglobin peptides and 33 unique DSSO inter-linked BSA peptides were identified.
These linkages were then mapped onto their corresponding protein linear sequences (
In the case of myoglobin, DSSO cross-linking identified several proximal helicase regions, such as α4-α5, α4-α7, α5-α8, α6-α8, and α5-310. In comparison, there is little overlap between DHSO and DSSO cross-link maps except the regions containing α4-α5 and α6-α8 (
In some embodiments, unlike myoglobin, DHSO and DSSO cross-linking of BSA have resulted in much more similar cross-linking profiles, meaning that similar interactions within BSA were identified (
The following Examples are non-limiting and other variants contemplated by one of ordinary skill in the art are included within the scope of this disclosure.
General chemicals were purchased from Fisher Scientific or VWR International. Bovine serum albumin (≥96% purity), myoglobin from equine heart (≥90% purity), and DMTMM (≥96% purity) were purchased from Sigma-Aldrich. Ac-SR8 peptide (Ac-SAKAYEHR (SEQ ID NO: 178), 98.22% purity) was custom ordered from Biomatik (Wilmington, Del.).
Disuccinimidyl sulfoxide (DSSO) was synthesized as previously published16. The 2-step synthesis scheme for DHSO from DSSO is depicted in
Synthetic peptide Ac-SR8 was dissolved in DMSO to 1 mM and cross-linked with DHSO in a 1:1 molar ratio of peptide to cross-linker in the presence of 1 equivalent of diisopropylethylamine and DMTMM. The resulting samples were diluted to 10 pmol/μL in 3% ACN/2% formic acid prior to MSn analysis.
50 μL of 50 μM BSA or 200 μM myoglobin in PBS buffer (pH 7.4) was reacted with DHSO in molar ratios of 1:5, 1:10, 1:20, and 1:30. The cross-linking reaction was initiated by adding equivalent concentrations of DHSO and DMTMM to protein solutions, reacted for 1 h at room temperature.
Cross-linked protein samples were subjected to either SDS-PAGE followed by in-gel digestion, or directly digested in solution. For in-gel digestion, cross-linked proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. The selected bands were excised, reduced with TCEP for 30 min, alkylated with iodoacetamide for 30 min in the dark, and then digested with trypsin at 37° C. overnight. Peptide digests were extracted, concentrated, and reconstituted in 3% ACN/2% formic acid for MSn analysis. For in-solution digestion, cross-linked proteins were first precipitated with TCA and then re-suspended in 8M urea buffer. Reduction and alkylation were performed prior to Lys-C/trypsin digestion as previously described24. The resulting digests were desalted using Waters C18 Sep-Pak cartridges and fractionated by peptide size exclusion chromatography (SEC) based on the protocol by Leitner et al.25. The fractions containing cross-linked peptides were collected for subsequent MSn analysis.
DHSO cross-linked peptides were analyzed by LC-MSn utilizing an Easy-nLC 1000 (Thermo Fisher, San Jose, Calif.) coupled on-line to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher, San Jose, Calif.) as previously described16,17. Each MSn experiment consists of one MS scan in FT mode (350-1400 m/z, resolution of 60,000 at m/z 400) followed by two data-dependent MS2 scans in FT mode (resolution of 7500) with normalized collision energy at 10% on the top two MS peaks with charges 4+ or higher, and three MS3 scans in the LTQ with normalized collision energy at 35% on the top three peaks from each MS2.
Monoisotopic masses of parent ions and corresponding fragment ions, parent ion charge states, and ion intensities from MS2 and MS3 spectra were extracted using in-house software based on the Raw_Extract script from Xcalibur v2.4 (Thermo Scientific) as previously described17. MS3 data was subjected to a developmental version of Protein Prospector (v.5.16.0) for database searching, using Batch-Tag against SwissProt.2014.12.4.random.concat databases limited to either the Bos taurus or Equus caballus taxonomy with mass tolerances for parent ions and fragment ions set as ±20 ppm and 0.6 Da, respectively. Trypsin was set as the enzyme with four maximum missed cleavages allowed. Cysteine carbamidomethylation was set as a constant modification. A maximum of four variable modifications were also allowed, including protein N-terminal acetylation, methionine oxidation, and N-terminal conversion of glutamine to pyroglutamic acid. In addition, three defined modifications representing cross-linker fragment moieties on aspartic acid and glutamic acid were selected: alkene (A, C3H4N2, +68 Da), sulfenic acid (S, C3H6N2SO, +118 Da), and unsaturated thiol (T, C3H4N2S, +100 Da) modifications. Initial acceptance criteria for peptide identification required a reported expectation value ≤0.1. The in-house program XL-Discoverer, a revised version of previously developed Link-Hunter, was used to validate and summarize cross-linked peptides based on MSn data and database searching16.
A novel approach to integration of multiplexed quantitation and different cross-linking chemistries for comprehensive structural comparison of protein complexes is provided. Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for elucidating architectures of protein complexes. Current XL-MS studies predominantly have used lysine targeting cross-linkers. However, there remains difficulty in characterizing interaction interfaces with limited lysine residues. Recently we developed an acidic residue targeting cross-linker, dihydrazide sulfoxide (DHSO)16, enabling improved coverage of protein interactions. As protein complexes are dynamic entities, which require quantitative XL-MS strategies for comparative analysis. To improve throughput and quantitation accuracy, a QMIX (Quantitation of Multiplexed Isobaric labelled cross-linked (X) peptides) strategy28 was developed for simultaneous comparison of multiple conformation states of protein complexes. The QMIX strategy was integrated with cross-linkers targeting different residues to dissect structural dynamics of human COP9 signalosome (CSN).
Two types of CSN complexes, CSN I and CSN II were purified, and cross-linked using DSSO or DHSO, respectively. The resulting cross-linked proteins were digested and cross-linked peptides were isolated using peptide SEC. Following SEC separation, selected fractions were labeled with TMT 6-plex™ isobaric Reagents (Thermo Scientific). The isobaric reagents labeled cross-linked peptides were mixed equally and analyzed by LC-MSn using an Easy-nLC 1200 (Thermo Scientific) coupled on-line to an Orbitrap Fusion Lumos Tribrid MS (Thermo Scientific). The resulting mass spectrometric data was subjected to protein database searching using Protein Prospector. Quantitation and cross-link identification was performed using in-house software package XL-Discoverer, designed to integrate various layers of MSn data to automatically summarize and validate the identification of cross-linking peptides.
CSN is a protein complex that functions as a deneddylase for cullin-RING ligases (CRLs) and thus modulates the assembly and action of CRLs. Although CSN is known to consist of eight subunits, a new stoichiometric subunit (i.e., CSN 9) has been recently identified16 that can enhance the CSN activity. To determine the molecular details underlying composition-dependent activation and action mechanisms of the CSN complex, we have established a new integrated strategy combining a multiplexed QXL-MS strategy (QMIX28) and the two sulfoxide-containing MS-cleavable cross-linkers with diverse functional groups, DSSO29 and DHSO30. The identification of cross-linked peptides is based on the XL-MS workflow designed for sulfoxide-containing MS-cleavable cross-linkers using multistage tandem mass spectrometry (MSn)28,16. Initial results from our XL-MS studies have identified 22 inter-subunit interactions comprised of 160 inter-subunit linkages using DHSO, and 19 inter-subunit interactions comprised of 107 inter-subunit linkages using DSSO. Additionally, 150 DSSO and 215 DHSO intra-subunit linkages were identified. Mapping of the linkages the CSN crystal structure (4D10), revealed that 84% of DSSO intra-links and 89% of DHSO intralinks were <30 Å. The detection of violated cross-links suggests an alternative positioning or unexpected flexibility in parts of the CSN complex. Concurrent usage of lysine and acidic residue targeting cross-linkers, has allowed for both validation of complimentary identified regions as well as elucidated regions where either lysine or acidic resides are sparse. The results have provided new structural details for refining the CSN structure and understanding its action mechanisms. The technologies presented here can be easily adapted to study other protein complexes.
Perspectives
In some embodiments, provided herein is the development and characterization of a new acidic residue-targeting, sulfoxide-containing MS-cleavable cross-linker, dihydrazide sulfoxide (DHSO), which is a new derivative of inventors' previously developed amine-reactive MS-cleavable reagent, DSSO16. In some embodiments, the extensive analyses herein have proven that DHSO cross-linked peptides possesses the same characteristics distinctive to peptides cross-linked by other sulfoxide-containing amine reactive cross-linkers16-18, thus enabling their simplified identification by MSn analysis. The unique features of DHSO will significantly facilitate cross-linking studies targeting acidic residues, which has been difficult in the past due to the large number of DIE present in protein sequences and complexity of their resulting cross-linked peptides for MS analysis.
Comparison of DHSO and DSSO cross-linking confirms the need of expanding the coverage of protein interactions using cross-linkers targeting different residues, especially when the distribution of specific amino acids is uneven. In some embodiments, this disclosure demonstrates the robustness and potential of the XL-MS technology based on sulfoxide-containing MS-cleavable cross-linkers and provides a viable analytical platform for the development of new MS-cleavable cross-linker derivatives to further define protein-protein interactions. Without being bound by any theory, it is believed that the development of these new tools will aid in the goal of understanding the structural dynamics of protein complexes and their mechanistic functions at the global scale in the future.
In some embodiments, the workflow disclosed herein will allow fast, effective, accurate and robust identification of DHSO cross-linked peptides. In some embodiments, the results herein demonstrate that DHSO cross-linked peptides possess the same characteristics distinctive to peptides cross-linked by other sulfoxide-containing amine reactive crosslinkers previously developed by the inventors, thus enabling their simplified identification by MSn analysis. The development of DHSO cross-linking will aid in the goal of defining protein-protein interactions at the global scale and understanding the structural dynamics of protein complexes and their mechanistic functions in cells.
As used herein, the section headings are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein.
In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting.
As used in this specification and claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
As used herein, “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
Although this disclosure is in the context of certain embodiments and examples, those skilled in the art will understand that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the embodiments and obvious modifications and equivalents thereof. In addition, while several variations of the embodiments have been shown and described in detail, other modifications, which are within the scope of this disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. It should be understood that various features and aspects of the disclosed embodiments can be combined with, or substituted for, one another in order to form varying modes or embodiments of the disclosure. Thus, it is intended that the scope of the present disclosure herein disclosed should not be limited by the particular disclosed embodiments described above.
Abbreviations
XL-MS: cross-linking mass spectrometry DSSO: disuccinimidyl sulfoxide
DMDSSO: dimethyl disuccinimidyl sulfoxide
Azide-A-DSBSO: Azide-tagged, Acid-cleavable DiSuccinimidyl BisSulfoxide
DHSO: dihydrazide sulfoxide, a.k.a. 3,3′-sulfinyldi(propanehydrazide)
MS: mass spectrometry
MS2: tandem mass spectrometry
MS3: third stage tandem mass spectrometry
MSn: multi-stage tandem mass spectrometry
CID: collisional induced dissociation
LC MSn: liquid chromatography multistage tandem mass spectrometry
Asp: aspartic acid
Glu: glutamic acid
All references cited in this disclosure are incorporated herein by reference in their entireties.
This application claims the benefit of U.S. Provisional Application No. 62/345,670, filed on Jun. 3, 2016, which is hereby incorporated by reference in its entirety.
This invention was made with Government support under Grant No. R01GM074830, R01GM074830-1052, and R01GM106003 awarded by the National Institutes of Health. The Government has certain rights in this invention.
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| Number | Date | Country | |
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| 20170350901 A1 | Dec 2017 | US |
| Number | Date | Country | |
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| 62345670 | Jun 2016 | US |
