Patent Application
20240353418
Accompanying this filing is a Sequence Listing entitled, “00058-078001.xml” created on Apr. 19, 2024 and having 4,613 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated by reference in its entirety for all purposes.
The disclosure provides for mass spectrometry (MS)-cleavable haloacetamide-based cross-linkers, and uses thereof, including for proteome-wide analysis of protein-protein interactions.
Protein-protein interactions (PPIs) are essential for the assembly of protein complexes, the active molecular modules for controlling cellular functionality and modulating physiological states. In recent years, cross-linking mass spectrometry (XL-MS) has been proven effective for studying PPIs and elucidating architectures of protein complexes in vitro and in vivo at the systems-level. Compared to other PPI methods, XL-MS enables the capture of endogenous PPIs without cell engineering. Identification of cross-linked peptides concurrently reveals PPI identities and interaction contacts at specific residues, which provide distance constraints defined by a given cross-linker to help refine existing structures and elucidate structures of protein complexes through computational modeling. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells.
The development of MS-cleavable cross-linkers have significantly advanced global PPI mapping to define the modularity and dynamics of cellular networks. This is due to the capability of MS-cleavable cross-linkers to provide simplified MS data for effective database searching, permitting cross-link identification with speed and accuracy. While successful, current proteome-wide XL-MS studies have all been relying on lysine-reactive cross-linkers. Although multiple cross-linking chemistries have been explored for XL-MS studies, lysine-reactive reagents remain the most popular. However, lysine-based XL-MS studies have only uncovered a fraction of proteomes. It is evident that additional cross-linking chemistries are important to expand PPI coverages and complement existing reagents. Provided herein are mass spectrometry (MS)-cleavable haloacetamide cross-linkers. While haloacetamides may have been shown to have slower cysteine reactivity compared with other cysteine interacting agents, they were found to be advantageous herein, in that they do not hydrolyze, are residue specific, and produce more homogenous cross-linked products for improved identification and quantitation. For the studies, a haloacetamide-based crosslinker was designed herein. This crosslinker, DBrASO (dibromoacetamide sulfoxide, aka, 3,3′-sulfinylbis(N′-(2-bromoacetyl) propanehydrazide) was generated by coupling bromoacetamide functional groups with sulfoxide-based MS-cleavability for unambiguous cross-link identification. It was demonstrated herein that DBrASO was an effective cysteine-reactive cross-linker for both simple and complex samples. Moreover, the disclosure provides for an innovative integrated DBrASO-based XL-MS analytical technique that couples a haloacetamide-based crosslinker of the disclosure with a newly developed two-dimensional fractionation method and LC-MSn data acquisition for complex PPI profiling. This technique has been successfully applied to cross-link HEK293 cell lysates, yielding a total of 11,478 unique cross-linked peptides describing an XL-proteome containing 2,297 proteins. The studies presented herein represent the first proteome-wide XL-MS study using a cysteine cross-linker. The results have demonstrated that a haloacetamide-based crosslinker of the disclosure (e.g., DBrASO) is effective for global XL-MS analysis and represents an attractive reagent to complement existing lysine-reactive cross-linkers for comprehensive PPI mapping at the systems-level.
In a particular embodiment, the disclosure provides a mass spectrometry (MS)-cleavable haloacetamide-based cross-linker comprising: one or more terminal haloacetamide groups; a single, centrally located sulfoxide group; and one or more MS-cleavable bonds; wherein the MS-cleavable haloacetamide-based cross-linker is configured to specifically interact with cysteine groups for mapping intra-protein interactions in a protein, or inter-protein interactions in a protein complex, or combinations thereof. In a further embodiment, the one or more MS-cleavable bonds is/are C—S bond(s) adjacent to the single, centrally located sulfoxide group. In yet a further embodiment, the mass spectrometry (MS)-cleavable haloacetamide-based crosslinker has 2 terminal haloacetamide groups that are located equal distant to the single, centrally located sulfoxide group. In another embodiment, the haloacetamide group has a structure of:
wherein, R1-R3 are individually selected from H, D and halo, wherein at least one of R1-R3 is a halo. In yet another embodiment, R1 is Br; and R2-R3 are H. In a further embodiment, the MS-cleavable haloacetamide-based cross-linker further comprises a first linker group, wherein one end of the first linker arm is connected to the single, centrally located sulfoxide group and the other end of the first linker group is connected to one of the terminal haloacetamide groups. In yet a further embodiment, the MS-cleavable haloacetamide-based cross-linker further comprises a second linker group, wherein one end of the second linker group is connected to the central sulfoxide group and the other end of the second linker group is connected to another terminal haloacetamide group, and wherein the first linker group and the second linker groups comprise identical functional groups and are symmetric. In a certain embodiment, the MS-cleavable haloacetamide-based cross-linker is DBrASO having the structure of:
In a particular embodiment, the disclosure also provides a mass spectrometry (MS)-cleavable haloacetamide-based cross-linker having the structure of Formula (I):
wherein, X1 and X2 are independently selected haloacetamide groups; L1 and L2 are independently selected linker groups; and wherein the dashed line indicates a MS-cleavable bond. In a further embodiment, X1 and X2 comprises the structure of
and wherein, R1-R3 are individually selected from H, D or halo, wherein at least one of R1-R3 is a halo. In yet a further embodiment, R1 is Br; and R2-R3 are H. In another embodiment, L1 and L2 are individually selected from an optionally substituted (C1-C12)alkyl, an optionally substituted (C1-C12)alkenyl,
wherein, z is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; z2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8; and n is an integer selected from 1, 2, 3, and 4. In yet another embodiment, L1 and L2 are selected from,
wherein, z1 is an integer selected from 0, 1, 2, 3, and 4; and z2 is an integer selected from 1, 2, 3, and 4. In a further embodiment, Lt and L2 are
wherein, z1 is an integer selected from 0, 1, 2 and 3; and z2 is an integer selected from 2, 3, and 4. In a certain embodiment, L1 and L2 are selected from
In a further embodiment, the MS-cleavable haloacetamide-based cross-linker has a structure of:
wherein, R1 is a halo selected from Cl, Br, and I. In a certain embodiment, the MS-cleavable haloacetamide-based cross-linker is DBrASO having the structure of:
In a particular embodiment, the disclosure also provides a method for mapping intra-protein interactions in a protein, inter-protein interactions in a protein complex, or any combination thereof, the method comprising: contacting the protein and/or the protein complex comprising a plurality of cysteine moieties with the MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 23 to form a cross-linked product; and digesting the cross-linked product to form a plurality of fragments, wherein a portion of the plurality of fragments comprises cross-linked peptide fragments; identifying and analyzing cross-linked peptide fragments using tandem mass spectrometry (MSn) to map intra-protein interactions in the protein and/or inter-protein interactions in the protein complex. In a further embodiment, a data-dependent MS3 acquisition method is used for identifying and analyzing the cross-linked peptide fragments.
In a certain embodiment, the disclosure further provides a method for mapping global protein-protein interactions (PPIs) from a sample comprising a plurality of proteins; contacting the sample comprising a plurality of proteins with the MS-cleavable haloacetamide-based cross-linker of claim 1 to form crosslinked proteins; digesting the crosslinked proteins to form crosslinked protein fragments or peptides; isolating fractions that are enriched with cross-linked protein fragments or peptides in the sample; analyzing the fractions using tandem mass spectrometry (MSn) and protein database searching to identify cross-linked protein fragments or peptides; and mapping the identified cross-linked protein fragments or peptides to generate a global structural map of PPIs. In a further embodiment, the fractions are isolated by using peptide size exclusion chromatography coupled with high pH reverse phase tip fractionation.
In a certain embodiment, the disclosure provides a composition, a method or a kit as substantially described herein.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a crosslinker” includes a plurality of such crosslinkers and reference to “the sulfoxide group” includes reference to one or more sulfoxide groups and equivalents thereof known to those skilled in the art, and so forth.
It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
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.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
The term “alkenyl”, as used in this disclosure, refers to an organic group that is comprised of carbon and hydrogen atoms that contains at least one double covalent bond between two carbons. Typically, an “alkenyl” as used in this disclosure, refers to organic group that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. While a C2-alkenyl can form a double bond to a carbon of a parent chain, an alkenyl group of three or more carbons can contain more than one double bond. It certain instances the alkenyl group will be conjugated, in other cases an alkenyl group will not be conjugated, and yet other cases the alkenyl group may have stretches of conjugation and stretches of nonconjugation. Additionally, if there is more than 2 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 3 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkenyl may be substituted or unsubstituted, unless stated otherwise.
The term “alkyl”, as used in this disclosure, refers to an organic group that is comprised of carbon and hydrogen atoms that contains single covalent bonds between carbons. Typically, an “alkyl” as used in this disclosure, refers to an organic group that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. Where if there is more than 1 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 2 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkyl may be substituted or unsubstituted, unless stated otherwise.
The term “alkynyl”, as used in this disclosure, refers to an organic group that is comprised of carbon and hydrogen atoms that contains a triple covalent bond between two carbons. Typically, an “alkynyl” as used in this disclosure, refers to organic group that contains that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. While a C2-alkynyl can form a triple bond to a carbon of a parent chain, an alkynyl group of three or more carbons can contain more than one triple bond. Where if there is more than 3 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 4 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkynyl may be substituted or unsubstituted, unless stated otherwise.
The term “aryl”, as used in this disclosure, refers to a conjugated planar ring system with delocalized pi electron clouds that contain only carbon as ring atoms. An “aryl” for the purposes of this disclosure encompasses from 1 to 4 aryl rings wherein when the aryl is greater than 1 ring the aryl rings are joined so that they are linked, fused, or a combination thereof. An aryl may be substituted or unsubstituted, or in the case of more than one aryl ring, one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof.
The term generally represented by the notation “Cx-Cy” (where x and y are whole integers and y>x) prior to a functional group, e.g., “C1-C12 alkyl” refers to a number range of carbon atoms. For the purposes of this disclosure any range specified by “Cx-Cy” (where x and y are whole integers and y>x) is not exclusive to the expressed range but is inclusive of all possible ranges that include and fall within the range specified by “Cx-Cy” (where x and y are whole integers and y>x). For example, the term “C1-C4” provides express support for a range of 1 to 4 carbon atoms, but further provides implicit support for ranges encompassed by 1 to 4 carbon atoms, such as 1 to 2 carbon atoms, 1 to 3 carbon atoms, 2 to 3 carbon atoms, 2 to 4 carbon atoms, and 3 to 4 carbon atoms.
The term “functional group” or “FG” refers to specific groups of atoms within molecules that are responsible for the characteristic chemical reactions of those molecules. While the same functional group will undergo the same or similar chemical reaction(s) regardless of the size of the molecule it is a part of, its relative reactivity can be modified by nearby functional groups. The atoms of functional groups are linked to each other and to the rest of the molecule by covalent bonds. Examples of FGs that can be used in this disclosure, include, but are not limited to, halogens, hydroxyls, anhydrides, carbonyls, carboxyls, carbonates, carboxylates, aldehydes, haloformyls, esters, hydroperoxy, peroxy, ethers, orthoesters, carboxamides, amines, imines, imides, azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosos, nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides, disulfides, sulfinyls, sulfos, thiocyanates, isothiocyanates, carbonothioyls, phosphinos, phosphonos, and phosphates.
The term “optionally substituted” refers to a functional group, typically a hydrocarbon or heterocycle, where one or more hydrogen atoms may be replaced with a substituent. Accordingly, “optionally substituted” refers to a functional group that is substituted, in that one or more hydrogen atoms are replaced with a substituent, or unsubstituted, in that the hydrogen atoms are not replaced with a substituent. For example, an optionally substituted hydrocarbon group refers to an unsubstituted hydrocarbon group or a substituted hydrocarbon group.
The term “substituent” refers to an atom or group of atoms substituted in place of a hydrogen atom. For purposes of this invention, a substituent would include deuterium atoms. Examples of substituents that can replace a hydrogen group in the structure of a crosslinker disclosed herein include, but are not limited to, halogen, hydroxyl, carboxyl, aldehyde, nitrile, isonitrile, nitro, amino, sulfide, alkyl (e.g., (C1-C6)alkyl), alkenyl (e.g., (C1-C6)alkenyls), alkynyl (e.g., (C1-C6)alkynyl), alkoxy (e.g., (C1-C6) alkoxy, ester (e.g., (C1-C6) ester), aryl, cycloalkyl, and heterocycle.
The term “substituted” with respect to hydrocarbons, heterocycles, and the like, refers to structures wherein the parent chain contains one or more substituents.
The term “unsubstituted” with respect to hydrocarbons, heterocycles, and the like, refers to structures wherein the parent chain contains no substituents.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, the exemplary methods and materials are disclosed herein.
All publications mentioned herein are incorporated by reference in full for the purpose of describing and disclosing methodologies that might be used in connection with the description herein. The publications are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.
Protein-protein interactions (PPIs) are essential for the assembly of protein complexes, the active molecular modules for controlling cellular functionality and modulating physiological states. In recent years, cross-linking mass spectrometry (XL-MS) has been proven effective for studying PPIs and elucidating architectures of protein complexes in vitro and in vivo at the systems-level. Compared to other PPI methods, XL-MS enables the capture of endogenous PPIs without cell engineering. Identification of cross-linked peptides concurrently reveals PPI identities and interaction contacts at specific residues, which provide distance constraints defined by a given cross-linker to help refine existing structures and elucidate structures of protein complexes through computational modeling. In particular, the development of MS-cleavable cross-linkers has significantly advanced global PPI mapping to define the modularity and dynamics of cellular networks. This is due to the capability of MS-cleavable cross-linkers to provide simplified MS data for effective database searching, permitting cross-link identification with speed and accuracy. While successful, current proteome-wide XL-MS studies have all been relying on lysine-reactive cross-linkers. Although multiple cross-linking chemistries have been explored for XLMS studies lysine-reactive reagents remain the most popular. However, lysine-based XL-MS studies have only uncovered a fraction of proteomes. It is evident that additional cross-linking chemistries are important to expand PPI coverages and complement existing reagents. In addition to lysine and acidic residues, cysteine is an attractive and unique amino acid owing to its critical importance in protein structure and function. Since the sulfhydryl group is highly reactive and can form disulfide bonds, cysteines modulate protein structures in multiple way including protein dimerization, metal coordination, redox regulation, and thermal stability. Cysteines can be labeled by many electrophilic reagents with high specificity and efficiency, which has been widely used in proteomic studies. While cysteine is one of the least abundant amino acids, it is often found at functional sites of proteins and its evolution appears to be highly regulated in proteins. Thus, cysteine cross-linking is expected to provide additional molecular details to help advance the understanding of protein interactions and structures. The homobifunctional MS-cleavable cysteine-reactive cross-linker bismaleimide sulfoxide (BMSO) has been used to define protein interactions and can be used in connection with lysine and acidic residue reactive reagents to expand PPI coverages. In addition, the multi-chemistry-based XL-MS approach was shown to facilitate the elucidation of the architectures of protein complexes by improving structural modeling with significantly increased precision. While maleimide chemistry has the fastest kinetics among the commonly used cysteine reactive groups, maleimide-labeled products can undergo retro-Michael addition and thiol exchange under physiological conditions. In addition, maleimides are prone to hydrolysis, which leads to the formation of ring-opened maleamic acids that are inactive to thiols. Finally, maleimide chemistry can be promiscuous, capable of labeling lysines at alkaline pH and resulting in unexpected complexity. Therefore, the exploration of alternative cysteine-labeling chemistries with higher specificity and less complexity would be advantageous to enable robust profiling of cysteine XL-proteomes for expansion of PPI mapping.
Provided herein are mass spectrometry (MS)-cleavable haloacetamide cross-linkers. While haloacetamides may have been shown to have slower cysteine reactivity compared with other cysteine interacting agents, they were found to be advantageous herein, in that they do not hydrolyze, are residue specific, and produce more homogenous cross-linked products for improved identification and quantitation. For the studies, a haloacetamide-based crosslinker was designed herein. This crosslinker, DBrASO (dibromoacetamide sulfoxide, aka, 3,3′-sulfinylbis(N′-(2-bromoacetyl) propanehydrazide) was generated by coupling bromoacetamide functional groups with sulfoxide-based MS-cleavability for unambiguous cross-link identification. It was demonstrated herein that DBrASO was an effective cysteine-reactive cross-linker for both simple and complex samples. Moreover, the disclosure provides for an innovative integrated DBrASO-based XL-MS analytical technique that couples a haloacetamide-based crosslinker of the disclosure with a newly developed two-dimensional fractionation method and LC-MSn data acquisition for complex PPI profiling. This technique has been successfully applied to cross-link HEK293 cell lysates, yielding a total of 11,478 unique cross-linked peptides describing an XL-proteome containing 2,297 proteins. The studies presented herein represent the first proteome-wide XL-MS study using a cysteine cross-linker. The results have demonstrated that a haloacetamide-based crosslinker of the disclosure (e.g., DBrASO) is effective for global XL-MS analysis and represents an attractive reagent to complement existing lysine-reactive cross-linkers for comprehensive PPI mapping at the systems-level.
In a particular embodiment, the disclosure provides a mass spectrometry (MS)-cleavable haloacetamide-based cross-linker comprising: one or more terminal haloacetamide groups; a single, centrally located sulfoxide group; and one or more MS-cleavable bonds; wherein the MS-cleavable haloacetamide-based cross-linker is configured to specifically interact with cysteine groups for mapping intra-protein interactions in a protein, or inter-protein interactions in a protein complex, or combinations thereof. In a further embodiment, the one or more MS-cleavable bonds is/are C—S bond(s) adjacent to the single, centrally located sulfoxide group. In another embodiment, fragmentation of the MS-cleavable haloacetamide-based cross-linker by mass spectrometry produces two major ion pairs. In yet another embodiment, the MS-cleavable haloacetamide-based cross-linker has little to no reactivity for lysine moieties. In a further embodiment, the MS-cleavable haloacetamide-based cross-linker has 1, 2, 3, 4, 5, 6, 7 or 8, or a range that includes, or is in between any two of the foregoing numbers, terminal haloacetamide groups. In a particular embodiment, the MS-cleavable haloacetamide-based cross-linker has 2 or 3 terminal haloacetamide groups. In a further embodiment, the MS-cleavable haloacetamide-based cross-linker has 2 terminal haloacetamide groups. In another embodiment, the mass spectrometry (MS)-cleavable haloacetamide-based crosslinker has 2 terminal haloacetamide groups that are located equal distant to the single, centrally located sulfoxide group. In yet another embodiment, the one or more terminal haloacetamide groups have a structure of:
wherein, R1-R3 are individually selected from H, D and halo, wherein at least one of R1-R3 is a halo. In a further embodiment, R1 is a halo group selected from I, Br, and Cl; and R2-R3 are H. In yet a further embodiment, R1 is Br. In a certain embodiment, the haloacetamide-based cross-linker is a homobifunctional cross-linker. In another embodiment, the mass spectrometry (MS)-cleavable haloacetamide-based cross-linker further comprises a first linker arm, wherein one end of the first linker group is connected to the single, centrally located sulfoxide group and the other end of the first linker group is connected to one of the terminal haloacetamide groups. In yet another embodiment, the mass spectrometry (MS)-cleavable haloacetamide-based cross-linker further comprises a second linker group, wherein one end of the second linker arm is connected to the single, centrally located sulfoxide group and the other end of the second linker arm is connected to another terminal haloacetamide group, and wherein the first linker group and the second group arm comprise identical groups and are symmetric. In yet another embodiment, the MS-cleavable haloacetamide-based cross-linker is DBrASO having the structure of:
In a particular embodiment, the disclosure provides a mass spectrometry (MS)-cleavable haloacetamide-based cross-linker having the structure of Formula (I):
wherein,
wherein, R1 is a halo selected from Cl, Br, and I. In yet another embodiment, the MS-cleavable haloacetamide-based cross-linker is DBrASO having the structure of:
The disclosure also provides methods or processes for mapping intra-protein interactions in a protein, inter-protein interactions in a protein complex, or any combination thereof. In a particular embodiment, the method comprises: contacting the protein and/or the protein complex comprising a plurality of cysteine moieties with the MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 23 to form a cross-linked product; digesting the cross-linked product to form a plurality of fragments, wherein a portion of the plurality of fragments comprises cross-linked peptide fragments; and identifying and analyzing cross-linked peptide fragments using tandem mass spectrometry (MSn) to map intra-protein interactions in the protein and/or inter-protein interactions in the protein complex. In a further embodiment, the MS-cleavable haloacetamide-based cross-linker is used at a molar ratio of 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or a range that includes or is between any two of the foregoing ratios (e.g., 1:5 to 5:1), to the protein and/or the protein complex. In a further embodiment, the MS-cleavable haloacetamide-based cross-linker is used at a molar ratio of 1:5 to 5:1 to the protein and/or the protein complex. In yet a further embodiment, the cross-linked product was digested with one or more proteases. Examples of proteases include, but are not limited to, serine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases. In a particular embodiment, the one or more proteases are serine proteases. In another embodiment, a data-dependent MS3 acquisition method is used for identifying and analyzing the cross-linked peptide fragments.
The disclosure also provides methods for mapping global protein-protein interactions (PPIs) from a sample comprising a plurality of proteins using a MS-cleavable haloacetamide-based cross-linker disclosed herein. In a particular embodiment, a method for mapping global protein-protein interactions (PPIs) from a sample comprising a plurality of proteins comprises: contacting the sample comprising a plurality of proteins with the MS-cleavable haloacetamide-based cross-linker of claim 1 to form crosslinked proteins; digesting the crosslinked proteins to form crosslinked protein fragments or peptides; isolating fractions that are enriched with cross-linked protein fragments or peptides in the sample; analyzing the fractions using tandem mass spectrometry (MSn) and protein database searching to identify cross-linked protein fragments or peptides; and mapping the identified cross-linked protein fragments or peptides to generate a global structural map of PPIs. In a further embodiment, the sample is a cellular sample. In yet a further embodiment, the cross-linked product was digested with one or more proteases. Examples of proteases include, but are not limited to, serine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases. In another embodiment, the one or more proteases are serine proteases. In yet another embodiment, the fractions are isolated by using peptide size exclusion chromatography coupled with high pH reverse phase tip fractionation. In a further embodiment, a data-dependent MS3 acquisition method is used for identifying the cross-linked protein fragments or peptides. In yet a further embodiment, the identified cross-linked protein fragments or peptide are mapped using various databases that profile protein to protein interactions. Examples of databases that profile protein to protein interactions, include, but are not limited to, APID, MiMI, iRefIndex, String, BioGrid, HPIDB, MINT, DIP, IntAct, HPRD, CORUM, and BioPlex. In yet another embodiment, the methods for mapping global protein-protein interactions (PPIs) further comprises the use of one or more additional MS-cleavable cross-linkers (e.g., BMSO, DSSO, DHSO, SDASO-S, Azide-A-DSBSO, Alkyne-A-DSBSO) with a MS-cleavable haloacetamide-based cross-linker disclosed herein.
Kits and articles of manufacture are also described herein. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.
For example, the container(s) can comprise one or more MS-cleavable haloacetamide cross-linkers disclosed herein, optionally in a composition or in combination with another agent as disclosed herein. The container(s) typically are made to exclude or limit light exposure for the contents of the container. Such kits optionally comprise a MS-cleavable haloacetamide cross-linker disclosed herein with an identifying description or label or instructions relating to its use in the methods described herein.
A kit will typically comprise one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint for use of a MS-cleavable haloacetamide-based cross-linker described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
A label can be on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. A label can be used to indicate that the contents are to be used for a specific analytical application. The label can also indicate directions for use of the contents, such as in the methods described herein.
The disclosure further provides that the methods and compositions described herein can be further defined by the following aspects (aspects 1 to 36):
1. A mass spectrometry (MS)-cleavable haloacetamide-based cross-linker comprising:
2. The MS-cleavable haloacetamide-based cross-linker of any one of aspect 1, wherein the one or more MS-cleavable bonds is/are C—S bond(s) adjacent to the single, centrally located sulfoxide group.
3. The MS-cleavable haloacetamide-based cross-linker of aspect 1 or aspect 2, wherein fragmentation of the MS-cleavable haloacetamide-based cross-linker by mass spectrometry produces two major ion pairs.
4. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 3, wherein the MS-cleavable haloacetamide-based cross-linker has little to no reactivity for lysine moieties.
5. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 4, wherein the MS-cleavable haloacetamide-based cross-linker has 1, 2, 3 or 4 terminal haloacetamide groups.
6. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 5, wherein the MS-cleavable haloacetamide-based cross-linker has 2 or 3 terminal haloacetamide groups.
7. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 6, wherein the MS-cleavable haloacetamide-based cross-linker has 2 terminal haloacetamide groups.
8. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 7, wherein the mass spectrometry (MS)-cleavable haloacetamide-based crosslinker has 2 terminal haloacetamide groups that are located equal distant to the single, centrally located sulfoxide group.
9. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 8, wherein the one or more terminal haloacetamide groups have a structure of:
10. The MS-cleavable haloacetamide-based cross-linker of aspect 9, wherein R1 is a halo group selected from I, Br, and Cl; and R2-R3 are H.
11. The MS-cleavable haloacetamide-based cross-linker of aspect 10, wherein R1 is Br.
12. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 11, wherein the haloacetamide-based cross-linker is a homobifunctional cross-linker.
13. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 12, wherein the mass spectrometry (MS)-cleavable haloacetamide-based cross-linker further comprises a first linker group, wherein one end of the first linker arm is connected to the single, centrally located sulfoxide group and the other end of the first linker group is connected to one of the terminal haloacetamide groups.
14. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 13, wherein the mass spectrometry (MS)-cleavable haloacetamide-based cross-linker further comprises a second linker group, wherein one end of the second linker arm is connected to the single, centrally located sulfoxide group and the other end of the second linker group is connected to another terminal haloacetamide group, and wherein the first linker arm and the second linker arm comprise identical groups and are symmetric.
15. A mass spectrometry (MS)-cleavable haloacetamide-based cross-linker having the structure of Formula (I):
wherein,
16. The MS-cleavable haloacetamide-based cross-linker of aspect 15,
17. The MS-cleavable haloacetamide-based cross-linker of aspect 16, wherein R1 is Br; and R2-R3 are H.
18. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 15 to 17, wherein L1 and L2 are individually selected from an optionally substituted (C1-C12)alkyl, an optionally substituted (C1-C12)alkenyl,
wherein, z1 is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; z2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8; and n is an integer selected from 1, 2, 3, and 4.
19. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 15 to 18, wherein L1 and L2 are selected from
wherein, z1 is an integer selected from 0, 1, 2, 3, and 4; and z2 is an integer selected from 1, 2, 3, and 4.
20. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 15 to 19, wherein L1 and L2 are
wherein, z1 is an integer selected from 0, 1, 2 and 3; and z2 is an integer selected from 2, 3, and 4.
21. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 15 to 20, wherein L1 and L2 are selected from
22. The MS-cleavable haloacetamide-based cross-linker any one of aspects 15 to 21, wherein L1 and L2 have a length less than 20 Å.
23. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 22, wherein the MS-cleavable haloacetamide-based cross-linker has a structure of:
24. The MS-cleavable haloacetamide-based cross-linker of any one of aspects 1 to 23, wherein the MS-cleavable haloacetamide-based cross-linker is DBrASO having the structure of:
25. A method for mapping intra-protein interactions in a protein, inter-protein interactions in a protein complex, or any combination thereof, the method comprising:
26. The method of aspect 25, wherein the MS-cleavable haloacetamide-based cross-linker is used at a molar ratio of 1:10 to 10:1 to the protein and/or the protein complex.
27. The method of aspect 26, wherein the MS-cleavable haloacetamide-based cross-linker is used at a molar ratio of 1:5 to 5:1 to the protein and/or the protein complex.
28. The method of any one of aspects 24 to 27, wherein the cross-linked product was digested with one or more proteases.
29. The method of aspect 28, wherein the one or more proteases are serine proteases.
30. The method of any one of aspects 24 to 29, wherein a data-dependent MS3 acquisition method is used for identifying and analyzing the cross-linked peptide fragments.
31. A method for mapping global protein-protein interactions (PPIs) from a sample comprising a plurality of proteins;
32. The method of aspect 31, wherein the sample is a cellular sample.
33. The method of any one of aspect 31 or aspect 32, wherein the cross-linked product was digested with one or more proteases.
34. The method of aspect 33, wherein the one or more proteases are serine proteases.
35. The method of any one of aspects 31 to 34, wherein the fractions are isolated by using peptide size exclusion chromatography coupled with high pH reverse phase tip fractionation.
36. The method of any one of aspects 34 to 35, wherein a data-dependent MS3 acquisition method is used for identifying the cross-linked protein fragments or peptides.
37. The method of any one of aspects 30 to 36, wherein the identified cross-linked protein fragments or peptide are mapped using various databases that profile protein to protein interactions.
The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.
Materials and Reagents. General chemicals were purchased from Fisher Scientific or VWR International. Bovine serum albumin (BSA) (≥96% purity) was purchased from Sigma-Aldrich. Sequencing-grade trypsin was purchased from Promega (Madison, WI). Ac-LR9 peptide (Ac-LADVCAHER (SEQ ID NO:1, 98% purity) was custom ordered from Biomatik (Wilmington, DE).
Synthesis and Characterization of DBrASO and Relevant Intermediates. The known bis-ester 2-S1 was treated with t-butyl carbazate to furnish the bis-protected hydrazide 2-S2. The protecting groups were removed upon treatment with TFA and the resulting salt was stirred with basic resin Amberlyst A21 to afford the free hydrazide 2-S3. To form the bromoacetamides, the bis-hydrazide was reacted with ester 2-S4 according to Trmčič et al. (Beilstein journal of organic chemistry 6:732-741 (2010)). The resulting bromoacetamide 2-S5 was finally oxidized from the sulfide to the sulfoxide to afford the cross-linker, DBrASO.
Bis(2,5-dioxopyrrolidin-1-yl) 3,3′-thiodipropionate (2-S1): To a solution of 3,3′-thiodipropionic acid (5.00 g, 28.1 mmol, 1.0 equiv), N-hydroxysuccinimide (12.9 g, 112 mmol, 4.0 equiv), and N,N-diisopropylethylamine (39.1 mL, 224 mmol, 8.0 equiv) in DMF (140 mL) at 0° C. was dropwise added to trifluoroacetic anhydride (15.8 mL, 112 mmol, 4.0 equiv). The orange solution was stirred for 2 h at 0° C., and then partitioned between EtOAc and brine. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brine (5×), dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting residue was purified via flash chromatography (40% EtOAc in CH2Cl2) to obtain NHS ester 2-S1 (8.21 g, 79%). 1H NMR (500 MHz, DMSO-d6): δ 3.01 (t, J=7.0 Hz, 4H), 2.87 (t, J=7.0 Hz, 4H), 2.82 (s, 8H); 13C NMR (126 MHz, DMSO-d6): δ 169.9, 167.6, 31.4, 25.6, 25.4.
Di-tert-butyl 2,2′-(3,3′-thiobis(propanoyl))bis(hydrazine-1-carboxylate) (2-S2): To a slurry of NHS ester 2-S1 (2.73 g, 7.33 mmol, 1.0 equiv) in CH2Cl2(38 mL) was added t-butyl carbazate (1.94 g, 14.7 mmol, 2.0 equiv) to obtain a clear orange solution. After stirring for 15.5 h at rt, the slurry was vacuum filtered to obtain a white flakey precipitate. The precipitate was washed with additional CH22 and then dried further in vacuo to obtain sulfide 2-S2 as a flakey white solid (2.28 g, 77%). Melting point: 187-190° C.; 1H NMR (500 MHz, DMSO-d6): δ 9.56 (s, 2H), 8.71 (s, 2H), 2.69 (t, J=7.4 Hz, 4H), 2.352 (t, J=7.0 Hz, 4H), 1.39 (s, 18H); 13C NMR (125 MHz, DMSO-d6): δ 170.1, 155.2, 79.0, 33.8, 28.1, 26.6; HRMS (ESI-TOF) m/z calculated for C16H30N4O6SNa (M+Na)+ 429.1779, found 429.1760.
3,3′-Thiodi(propanehydrazide) (2-S3): Five separate vials each containing a solution of sulfide 2-S2 (200 mg, 0.492 mmol, 1.0 equiv) in trifluoroacetic acid (1.0 mL) was stirred at rt for 16 h and then concentrated in vacuo. The resulting residue from each vial was dissolved in a 1:1 solution of MeOH:CH2Cl2 and added to a mixture of Amberlyst A21 resin (4.0 g, 10.0 equiv by mass of salt product) in CH2Cl2. The mixture was stirred for 45 min at rt, after which the resin was filtered and washed with a 1:1 solution of MeOH:CH2Cl2. The filtrates were combined and concentrated in vacuo to obtain hydrazide 2-S3 as a tan solid (293 mg, 58%). 1H NMR (600 MHz, DMSO-d6): δ 9.00 (s, 2H), 4.34 (s, br, 4H), 2.67 (t, J=7.3 Hz, 4H), 2.28 (t, J=7.3 Hz, 4H); 13C NMR (151 MHz, DMSO-d6): δ 169.9, 33.8, 26.9; HRMS (ESI-TOF) m/z calculated for C6H15N4O6S (M+H)+ 207.0911, found 207.0908.
2,5-Dioxopyrrolidin-1-yl 2-bromoacetate (2-S4): To a solution of N-hydroxysuccinimide (1.32 g, 11.5 mmol, 1.0 equiv) in CH2Cl2 (15.0 mL) was dropwise added NEt3 (1.92 mL, 13.8 mmol, 1.2 equiv) and a solution of bromoacetyl bromide (1.0 mL, 11.5 mmol, 1.0 equiv) in CH2Cl2 (15.0 mL) at 0° C. After stirring at 0° C. for 1 h, the reaction was quenched with saturated NaHCO3 solution. The organic phase was washed with a saturated NaHCO3 solution (2×), 1 M HCl (3×), and brine, dried over Na2SO4, and concentrated in vacuo to obtain NHS ester 2-S4 as a tan solid (1.95 g, 72%). 1H NMR (500 MHz, CDCl3): δ 4.10 (s, 2H), 2.86 (s, 4H); 13C NMR (126 MHz, CDCl3) δ 168.6, 163.1, 25.7, 21.3.
3,3′-Thiobis(N′-(2-bromoacetyl)propanehydrazide) (2-S5): To a solution of hydrazide 2-S3 (293 mg, 1.42 mmol, 1.0 equiv) in H2O (1.0 mL) was added a slurry of NHS ester 2-S4 (671 mg, 2.84 mmol, 2.0 equiv) in MeCN (1.0 mL). The vial containing ester 1-25 was washed with a MeCN:H2O solution (1 mL) and the slurry was added to the reaction vial. After stirring at rt for 25 min the precipitate was vacuum filtered and washed with a 1:1 solution of MeCN:H2O to obtain bromoacetamide 2-S5 as an off white powder (333 mg, 52%). Melting point: 166-170° C.; 1H NMR (500 MHz, DMSO-d6): δ 10.38 (s, 2H), 10.12 (s, 2H), 3.91 (s, 4H), 2.72 (t, J=7.3 Hz, 4H), 2.43 (t, J=7.2 Hz, 4H); 13C NMR (126 MHz, DMSO-d6): δ 168.9, 164.5, 33.6, 27.1, 26.6; HRMS (ESI-TOF) m/z calculated for C10H16Br2N4O4SNa (M+Na)+ 468.9152, found 468.9170.
3,3′-Sulfinylbis(N′-(2-bromoacetyl)propanehydrazide) (DBrASO): To a solution of sulfide 2-S5 (400 mg, 0.893 mmol, 1.0 equiv) in HFIP (4.5 mL) at rt was added 30% aqueous H2O2 (0.18 mL, 1.78 mmol, 2.0 equiv). The tan slurry became clear and after stirring for 25 min at rt the reaction was slowly quenched with DMS (0.20 mL, 2.70 mmol, 3.0 equiv). A white solid precipitated out of solution as the DMS was added, and the slurry was stirred for an additional 10 min. The reaction mixture was vacuum filtered and the white precipitate was further dried in vacuo to obtain DBrASO as a white powder (235 mg, 57%). Melting point: 175-178° C.; 1H NMR (600 MHz, DMSO-d6): δ 10.38 (s, 2H), 10.22 (s, 2H), 3.92 (s, 4H), 3.09-3.02 (m, 2H), 2.86-2.79 (m, 2H), 2.58 (t, J=6.8 Hz, 4H); 13C NMR (151 MHz, DMSO-d6): δ 168.6, 164.7, 46.1, 27.0, 25.8; HRMS (ESI-TOF) m/z calculated for C10H16Br2N4O5SNa (M+Na)+ 484.9101, found 484.9118.
DBrASO Cross-linking of Synthetic Peptide. Synthetic peptide Ac-LR9 was dissolved in DMSO to a final concentration of 1 mM. DBrASO was added at 1:1 molar ratio to the peptide. The reaction was carried out at room temperature for one hour in the dark. The resulting samples were diluted to 10 pmol/μL in 3% ACN/2% formic acid prior to LC-MSn analysis.
DBrASO Cross-linking of Bovine Serum Albumin and Cell lysates. 50 μL of 50 μM BSA in PBS buffer (2 mM TCEP, pH 7.6) was reacted with DBrASO at room temperature for 1 h in the dark. The HEK 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin until 90% confluence. After washing with PBS for three times, cell pellets were resuspended in lysing buffer (100 mM sodium chloride, 50 mM sodium phosphate, 10% glycerol, 2 mM ATP, 1 mM TCEP, 10 mM MgCl2, 1×protease inhibiter (Roche), 1× phosphatase inhibitor, and 0.5% NP-40, pH 7.6). The cell debris were removed by centrifugation at 13,000 rpm for 15 min at 4° C. The supernatant was collected and adjusted to 1 mg/mL, followed by cross-linking with 10 mM DBrASO at room temperature for 1 h in dark.
Cross-linked Protein digestion. After cross-linking, the samples were transferred onto a 30 KDa centrifugal filter and washed with 8M urea to remove excess linker. The denatured proteins were reduced with 2 mM TCEP for 30 min and alkylated with 10 mM iodoacetamide for 30 min in the dark. After washing with 25 mM ammonium bicarbonate (ABC), the sample was resuspended in 8M urea/25 mM ABC buffer, and Lys-C (enzyme to protein ratio of 1:100) was added to the solution. The resulting mixture was incubated at 37° C. for 4 h. The concentration of urea was then reduced to 1.5 M for trypsin digestion (enzyme to protein ratio of 1:50) at 37° C. overnight. The digested peptides were acidified by adding 1% TFA to final concentration of 0.1% and desalted by Sep-Pak C18 cartridge prior to LC-MSn analysis.
SEC-HpHt Fractionation of Cross-linked Peptides. Cross-linked peptides of cell lysates were further separated by peptide size exclusion chromatography coupled with high pH reverse phase tip fractionation (SEC-HpHt) as described in Jiao et al. (Analytical Chemistry 94:4236-4242 (2022)). Briefly, 250 μg of desalted peptides was dissolved in 30% ACN/0.1% FA, then loaded onto a Superdex™ Peptide 3.2/300 SEC column for separation. The two SEC fractions containing cross-linked peptides were collected, dried, dissolved in 160 μL of ammonia water (pH 10) and subjected to HpHt fractionation separately.
For HpHt separation, a pipette tip (200 μL) was first blocked with a layer of C8 membrane (Empore 3M). After filling with 5 mg of C18 solid phase (3 m, Durashell, Phenomenex). the tip was balanced with 90 μL of methanol, 90 μL of ACN and 90 μL of ammonia water (pH 10) sequentially. Then, dissolved peptides were loaded onto the tip and centrifuged at 1,200 rpm until the liquid level was close to the beads. After washing with another 90 μL of ammonia water (pH 10) for desalting, the peptides were eluted with increasing percentage of ACN in ammonia water (6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, 35%, and 50%). The 25%, 30%, 35% and 50% fractions were combined to 6%, 9%, 12% and 21% fractions, respectively. The final 6 fractions were vacuum dried and stored at −80° C. before LC-MSn analysis.
LC-MSn analysis. Cross-linked peptides were analyzed by LC-MSn using an UltiMate 3000 RSLC coupled with an Orbitrap Fusion Lumos mass spectrometer. Samples were loaded onto a 50 cm×75 m Acclaim PepMap C18 column and separated over a 120 min gradient of 4% to 25% acetonitrile at a flow rate of 300 nL/min. The top 4 data-dependent MS3 acquisition method was used for the identification of DBrASO cross-linked peptides. Ions with charge of 4+ to 8+ in the MS1 scan were selected for MS2 analysis. The top 4 most abundant fragment ions in MS2 scan were further fragmented by CID with a collision energy of 35%.
Identification of Cross-linked Peptides. Raw data were converted to mgf files by MSConvert (Protein Wizard 3.0.21288). Extracted MS3 spectra was subjected to Protein Prospector (v.6.3.3) for database searching (using Batch-Tag against SwissProt.2019.04. 08 random concatenated database). The mass tolerances were set as ±20 ppm for parent ions and 0.6 Da for fragment ions. Trypsin was set as the enzyme with three maximum missed cleavages allowed. A maximum of four variable modifications were also allowed, including cysteine carbamidomethylation, methionine oxidation, N-terminal acetylation, and N-terminal conversion of glutamine to pyroglutamic acid. Three defined DBrASO cross-linked modification on cysteine, including alkene (C5H6O2N2, +126 Da), thiol (C5H6O2N2S, +158 Da) and sulfenic acid (C5H8O3N2S, +176 Da) were also added as variable modifications. MSn data were integrated via in-house software xl-Tools to identify cross-linked peptide pairs.
PPI Analysis and Structural Mapping. The PPI network of HEK293 cell lysates was derived based on DBrASO XL-MS data. BioPlex (bioplex.hms.harvard.edu/), BioGrid (thebiogrid.org/) and CORUM (mips.helmholtz-muenchen.de/corum) were used to determine protein complexes. Gene Ontology enrichment was performed using the R package “ClusterProfiler”. Cross-links mapping to high-resolution structures of protein complexes was performed as described in Wheat et al. (Proceedings of the National Academy of Sciences 118 (2021).
Design and Synthesis of a Novel MS-Cleavable Homobifunctional Cysteine Cross-Linker. To improve the specificity and homogeneity of cysteine cross-linking, an alternative sulfhydryl chemistry, i.e., the haloacetamide group was utilized to create a novel sulfoxide-containing MS-cleavable cysteine-reactive homobifunctional cross-linker (see
MS Characterization of DBrASO Cross-Linked Peptides. While DBrASO cross-linking is expected to result in different types of cross-linked peptides, the focus was on the characterization of DBrASO interlinked peptides due to their importance in delineating protein interactions and structures. Like other residue-specific sulfoxide-containing crosslinkers, such as DSSO and BMSO, cleavage of either MS-cleavable C—S bond physically separates the two crosslinked peptide constituents, yielding peptide fragment ion pairs (i.e., αA/βS or αS/βA). These resulting peptide fragments are modified with alkene (A) or sulfenic acid (S) moieties, remnants of DBrASO following collision-induced dissociation (CID). The sulfenic moiety typically undergoes dehydration to become a more stable and dominant unsaturated thiol (T) moiety. Therefore, the two dominant fragment pairs for a DBrASO cross-linked peptide α-β are expected to be αA/βT and αT/βA (see
The fragmentation characteristics of DBrASO cross-linked peptides were first evaluated using a cysteine-containing synthetic peptide Ac-LR9 (i.e., Ac-LADVCAHER). MS2 analysis of the DBrASO cross-linked Ac-LR9 homodimer (m/z 603.76984+) resulted in three dominant fragments: αA (m/z 591.23792+), αT (m/z 607.25932+), and αS (m/z 616.26542+), with the alkene- and thiol-modified peptides being most dominant as anticipated (see
DBrASO XL-MS Analysis of BSA. To assess the efficiency of DBrASO for protein cross-linking, the readily available protein bovine serum albumin (BSA) was crosslinked due to its high cysteine content and common use in XL-MS studies. To characterize DBrASO cross-linking of BSA, LC-MSn analysis was employed to identify DBrASO cross-linked peptides. As an example, MSn analysis of a representative DBrASO interlinked peptide α-β of BSA (m/z 676.55224+) is illustrated in
To examine whether the differences in cysteine-labeling efficiency between haloacetamide and maleimide functional groups could impact BSA cross-linking, BMSO cross-linking on BSA was performed using the same conditions as DBrASO. A total of 75 BMSO C-C linkages were identified from three biological replicates, and 92% of them were considered satisfactory (≤45 Å) (see
DBrASO XL-MS Analysis of HEK293 Cell Lysates. Recent advances in XL-MS technologies have successfully enabled proteome-wide XL-MS studies using lysine-reactive cross-linkers. However, other cross-linking chemistries which complement lysine-reactive reagents in proteome-wide PPI mapping have not yet been investigated. Previous XL-MS analyses of proteins and protein complexes have proven the benefits of using multiple cross-linking chemistries to expand PPI mapping and enhance the precision of structural modeling. To explore the feasibility of cysteine-reactive cross-linkers for proteome-wide XL-MS studies, DBrASO cross-linking of HEK293 cell lysates were performed (see
Comparisons of DBrASO and DSSO XL-Proteomes. To illustrate the differences between cysteine and lysine crosslinking chemistries, the identified 2297 DBrASO XL-MS proteome was compared with a previously published DSSO XL proteome of HEK293 cell lysates. Similar to DSSO, DBrASO cross-linked proteins were enriched in various subcellular compartments, including the nucleus, cytosol, and mitochondria (see
The abundance distribution of XL proteomes were plotted using the intensity-based absolute quantification (iBAQ) values of the HEK293 MS proteome in ProteomicsDB. 38 Out of the 2297 DBrASO-identified proteins, 2152 were correlated with iBAQ values, and their distribution indicated that high-abundance proteins were well-represented in the cysteine XL-proteome (see
Examination of Cross-Links by Structural Mapping. The DBrASO XL-proteome of HEK293 cell lysates is composed of 2297 proteins, in which 975 CORUM protein complexes were found and 74.5% of them were identified with more than 50% protein composition of each protein complex. Based on the number of C-C linkages, the most representative complexes are the cytoplasmic ribosome, CCT, 26S proteasome, and pre-mRNA spliceosome complexes. To assess the validity of DBrASO cross-links, a total of 1380 C-C linkages (879 intraprotein and 501 interprotein) were mapped onto available high-resolution structures of 126 protein complexes. While 717 out of the 879 intraprotein C-C linkages (81.6%) corresponded to Cα-Cα distances below the DBrASO threshold (≤40 Å), the majority of the 501 interprotein C-C linkages were found to be nonsatisfactory. However, it is noted that 97% of the violating interprotein cross-links were determined to be associated with ribosomal proteins (460/475). This is not surprising as the ribosomal complex is known to be highly dynamic, and various lysine-based XL-MS data sets of purified samples and cell lysates have revealed many nonsatisfactory interprotein cross-links from ribosomal proteins. Therefore, DBrASO XL data further support the structural plasticity of the ribosomal complex.
A well-represented complex in the data set is the eight subunit CCT complex, identified with 153 C-C cross-links describing 148 intra-subunit and five intersubunit interactions involving all eight subunits. Out of the 145 C-C linkages that were able to be mapped onto the CCT complex structure (PDB: 7LUP), the majority (74%) were satisfied with the Ca-cross-link satisfaction was determined to be 91% (40/44) and 86% (37/43), respectively (see
DBrASO XL-PPI Network of HEK293 Cell Lysates. To visualize the identified PPIs, an XL-PPI network comprising 505 nodes and 856 edges was generated, denoting 856 interprotein interactions (see
Among the novel PPIs, 128 correspond to interactions involving ribosomal proteins. For instance, multiple interaction contacts between UBA52 and 40S/60S ribosomal proteins were uniquely identified by DBrASO cross-linking but not by lysine-reactive reagents. This is due to the fact that the N-terminus of UBA52 (also called ubiquitin-60S ribosomal protein L40) has identical sequence to ubiquitin (1-76 AA). Thus, any cross-linked peptides involving the N-terminus of UBA52 (1-76) would be ambiguous as they could belong to ubiquitin and/or other ubiquitin precursors. While multiple lysines exist at the C-terminus (77-128 AA) of UBA52, crosslinked peptides involving the C-terminus have not been reported using lysine-reactive cross-linkers. Here, cysteine XL by DBrASO produced physical evidence to support the direct interaction of UBA52 with ribosomal proteins due to the identification of its unique C-terminal peptide (115CGHTNNLRPK124) (SEQ ID NO:4). Additionally, 34 novel interactions identified here are related to GAPDH, including its interactions with heat shock proteins, 40S ribosomal, 60S ribosomal, TRiC-complex, and other proteins (see
A number of embodiments have been described herein. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.
This application claims priority under 35 U.S.C. § 119 from Provisional Application Ser. No. 63/461,231, filed Apr. 21, 2023 the disclosure of which is incorporated herein by reference.
This invention was made with Government support under Grant Nos. R01GM074830 and R01GM130144, awarded by the National Institutes of Health. The Government has certain rights in the invention
| Number | Date | Country | |
|---|---|---|---|
| 63461231 | Apr 2023 | US |
