PROTEIN INTERACTION REPORTER AGENTS AND METHODS FOR USING SAME
Particular aspects provide novel protein interaction reporter (PIR) compounds (e.g., formulas I and II), comprising at least two protein reactive moieties (e.g., N-hydroxysuccinamide), each linked to a reporter moiety (e.g., mass reporter) by a covalent labile bond that is differentially cleavable with respect to peptide bonds (e.g., by a method such as collisional activation in a mass spectrometer, activation by electron capture dissociation (ECD), photoactivation, etc.), wherein the reporter moiety is operatively releasable from the PIR agent upon cleavage of the labile bonds, the released reporter moiety having a characteristic identifying property or label (e.g., m/z value). Particular PIRs comprise a mass reporter moiety, and further comprise an affinity group, (e.g., biotin), linked to the PIR (e.g., to the mass reporter moiety) by a selectively cleavable bone (e.g. photo-labile bond)). Additional aspects provide methods for characterizing intermolecular or intramolecular protein interactions using one or more inventive PIR compounds.
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This application is a continuation of U.S. patent application Ser. No. 11/282,403 (now issued as U.S. patent), filed 18 Nov. 2005 and entitled PROTEIN INTERACTION REPORTER AGENTS AND METHODS FOR USING SAME, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 60/629,396, filed 18 Nov. 2004 and entitled CHEMICAL CROSS-LINKERS, METHODS OF USING CHEMICAL CROSS-LINKERS, AND METHODS FOR STUDYING PROTEIN-PROTEIN INTERACTIONS, both of which are incorporated by reference herein in their entirety.
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCHThis work was supported by U.S. Department of Energy Grant No. DE-FG02-04ER63924, and NIH grant No. S10 RR017805-01. The United States has certain rights in this invention, pursuant to 35 U.S.C. §202(c)(6).
FIELD OF THE INVENTIONParticular aspects relate generally to protein or polypeptide cross-linking and methods for studying same, and more particularly to novel, substantially useful cross-linking compounds and methods for using same.
BACKGROUNDMany cellular functions are carried out through large multi-protein complexes, rather than individual proteins. The systematic mapping of proteome-wide protein interactions to produce a comprehensive network of protein-protein interactions, or ‘interactome,’ is thus essential for understanding processes in biological systems. However, characterization of protein interactions is challenging because most protein interactions are transient, and many are unstable. In addition, multi-protein complexes possess no common factors or physical properties that can be used as an analytical handle.
Nonetheless, numerous efforts have been undertaken to profile large-scale protein interaction networks within a given cell, as the study of protein-protein interactions is a critical component in systems biology research directed at characterizing complex network interactions and behavior to better enable comprehension of protein function. Two popular methods to study protein-protein interactions are: (i) the yeast two-hybrid system, which is a genetic approach; and (ii) the use of protein cross-linking in combination with mass spectrometry (MS) to analyze purified protein complexes as well as their topological structures.
Protein cross-linking approaches can, at least in principal, not only identify what proteins interact within a complex, but can also pinpoint where proteins interact. However, despite much interest in profiling protein-protein interactions using cross-linking strategies and the availability of many commercial cross-linkers, (see, e.g., Pierce Chemicals, Double-Agents Cross-Linking Guide, 1999) few reports have illustrated progress in application/implementation of cross-linker approaches on a proteome-wide scale. This limitation is primarily due to inherent complexity in cross-linking reaction mixtures, which include large amounts of undesirable or unexpected products (see, e.g., Swaim, C. L. et al., J Am Soc Mass Spectrom 15:736-749, 2004), in addition to 1:1 linkage of interacting proteins. Moreover, the challenge of interpreting cross-linking approach results derives not only from the complexity of cross-linking reaction and digestion mixtures, but also from the complexity of MS/MS mass spectra—typically used for investigation of cross-linked peptides.
Therefore, there is a pronounced need in the art for novel cross-linking agents, and methods for using such agents, to simplify the identification and characterization of cross-linked proteins and peptides and thus facilitate the viability and utility of mass spectrometry for effective ‘interactome’ characterization.
SUMMARYParticular aspects provide novel multivalent protein interaction reporter (PIR) compounds, and methods for using same (e.g., mass spectrometry-based methods, etc., as described in the working examples herein).
Additional aspects provide novel protein interaction reporter (PIR) compounds having formulas I or II:
where R1, R2, W, X, Y, Z and U are as defined herein below.
The inventive PIR compounds comprise at least two protein reactive moieties (e.g., N-hydroxysuccinamide), each linked to a reporter moiety (e.g., mass reporter) by a covalent labile bond that is differentially cleavable with respect to peptide bonds (e.g., by a method such as collisional activation in a mass spectrometer, activation by electron capture dissociation (ECD), photoactivation, etc.), wherein the reporter moiety is operatively releasable from the PIR agent upon cleavage of the labile bonds, the released reporter moiety having a characteristic identifying property or label (e.g., m/z value). Particular PIRs comprise a mass reporter moiety, and further comprise an affinity group, (e.g., biotin), linked to the PIR (e.g., to the mass reporter moiety) by a selectively cleavable bone (e.g. photo-labile bond)).
Additional aspects provide a method of characterizing intermolecular or intramolecular protein interactions, comprising: obtaining a sample comprising at least one protein; contacting the at least one protein with at least one multivalent protein interaction reporter (PIR) compound, under conditions suitable for cross-linking to provide a cross-linked protein sample, wherein the PIR compound comprises at least two protein reactive moieties, each bound to a shared characteristic reporter moiety by a covalent labile bond, wherein the labile bonds can be differentially cleaved with respect to peptide bonds, wherein the reporter moiety is operatively releasable from the PIR agent upon differential cleavage of the labile bonds to provide for a characteristic released reporter moiety; differentially cleaving the labile bonds to provide for a released reporter moiety; and subjecting the cleaved products to an analysis suitable to distinguish the cleavage products by virtue of being the reporter moiety or by inclusion of a protein reactive moiety or portion thereof, wherein characterizing intermolecular or intramolecular protein interaction or structure is, at least in part, afforded. In particular aspects, the method of further comprises digesting, prior to differential bond cleavage, the cross-linked protein sample with at least one protease.
Particular aspects relate to compositions and methods having substantial utility for characterizing inter-molecular or intra-molecular protein interactions or structures, utilizing novel multivalent protein interaction reporter (PIR) compounds. Certain embodiments relate to methods comprising use of PIR compounds to cross-link proteins or peptides, followed by subsequent analysis by mass spectrometry (MS). Upon exposure to a sufficient amount of activation energy in a mass spectrometer, PIR compounds undergo labile bond cleavage that produces a characteristic reporter ion with a distinct, identifiable mass to charge (m/z) ratio. The reporter ion serves to identify fractions that have cross-linked peptides, even where such fractions are present as a small percentage of a large, complex mixture of proteins and cross-linkers. Generally, the amount of energy required to release the reporter ion is lower than the amount of energy required to break a peptide bond. Following the release of the reporter ion, the peptides that were cross-linked by an inventive PIR compound may be subjected to one or more additional rounds of MS to determine the identity and/or sequence of the peptide.
Exemplary PIR compounds (
The at least two protein reactive groups may be identical or different, and are chemical moieties that form covalent links with proteins and/or peptides. Certain embodiments of the invention utilize an N-hydroxysuccinamide (NHS) ester as a protein reactive group. NHS esters generally react with amine groups of proteins or peptides. Other examples of protein reactive groups encompassed herein include aryl azides (react with primary amines), carbodiimides (react with amines or carboxyls), hydrazide (reacts with carbohydrates), hydroxymethyl phospine (reacts with amines), imidoesters (react with amines), isocyanates (react with hydroxyls), maleimides (react with sulfhydryls), PFP esters (react with amines), psoralen (a photoreactive intercalator that reacts with thymine), vinyl sulfones (react with sulfhydryls, amines, and hydroxyls), and pyridyl disulfides (react with sulfhydryls).
Inventive PIR compounds may have two or more protein reactive groups. Inventive PIR compounds are referred to as “multivalent,” because PIR compounds may form cross-links with more than one protein or peptide. Additionally, the protein reactive groups in a single PIR compound may all be the same, or alternatively, two or more different protein reactive groups may be present in a single PIR compound. It will be apparent to one of ordinary skill in the art that the choice of protein reactive groups may be strategically selected for particular purposes, and the results compared between and among analyses using different protein reactive groups.
“Cleavage groups” refer to regions of the inventive PIR compounds that comprise a labile covalent bond, cleavable under appropriate conditions to separate the protein reactive groups from the mass reporter group, which is situated between two protein reactive groups. Generally, cleavage groups are located between the protein reactive groups and the reporter group. As used herein, the term “labile covalent bond” refers to a bond that may be broken under conditions that generally do not result in the breaking of peptide bonds.
Certain embodiments of the invention comprise cleavage groups that are “MS-labile”. MS-labile bonds are bonds that generally break during mass spectrometry conditions wherein relatively low activation energy is used. Under these conditions, the inventive PIR molecules fragment at the cleavage groups (at the labile bonds), while the peptide bonds of the cross-linked proteins generally remain intact.
Chemical moieties that may be used as cleavage groups in PIR compounds include, but are not limited to N-functionalized heterocyclic aromatic compounds, such as substituted or unsubstituted indole or pyridyl-based compounds, secondary and tertiary amines wherein one bond is to a carbon atom neighboring group with significant π-delocalization, such as substituted biphenyl compounds, dithiols, phosphates, and metal ligand complexes. For PIR compounds having more than one cleavage group, the cleavage groups may all be the same, or they may be different (e.g., two or more different cleavage groups.
Certain embodiments of the invention relate to the use of “Rink” or Rink-related compounds as cleavage groups. The term “Rink” refers to a trialkoxy-diphenyl-methylester disclosed by Rink et al. (see, e.g., Rink, H.; Tetrahedron Lett. 28:3787-3790, 1987). Additionally, U.S. Pat. No. 5,124,478 (incorporated herein by reference in its entirety) discloses a number of Rink-related compounds that have MS-labile bonds and may be useful as cleavage groups of the inventive PIR compounds. Additional embodiments of the invention comprise indole-based MS-labile cleavage groups.
The reporter group of a PIR compound may also be referred to as a “central mass reporter moiety,” and is a region of the molecule in between the cleavage points defined by the cleavage groups. Generally, when labile covalent bonds are broken at both cleavage groups, a reporter group is released. The reporter group, upon release from the PIR compound, has a characteristic property that identifies it as the reporter group (e.g., a characteristic m/z value, etc.).
PIR compounds may further comprise an affinity group or tag that is covalently linked to the reporter group (e.g., via a connector region or arm). Such affinity tags may be used to react with or bind to a recognition element, which may be free in solution or immobilized on a solid. The affinity tag may be used to at least partially separate PIR compounds which may be cross-linked to peptides or proteins from other proteins that are not cross-linked to PIR compounds. Certain embodiments of the invention comprise biotin as an affinity tag that is attached to a reporter group (e.g., via an hydrophilic connector arm). Other affinity tags or reactive groups useful for this purpose include, but are not limited to poly histidine (4-14, preferably 6-10 residues), antibodies or antigens, benzophenone, sulfhydryl groups, and substituted or unsubstituted aryl azide. Examples of affinity capture components include, but are not limited to biotin and avidin, an antibody and an antigen, an aptamer and a small molecule, a polyhistidine tag and nickel, and a reactive group such as a thiol, which can undergo a Michael addition.
The connector region or arm between the affinity group and the reporter group may vary depending on the proposed application of the PIR compound. The connector regions or arms may vary in length, and, for example, may be hydrophobic or hydrophilic. Hydrophilic connector regions or arms may be selected to promote solubility and facilitate interactions between proteins at their physiological pH. Certain embodiments of the invention provide connector regions or arms that are derived from polyethylene glycol. However, other connector regions or arms, such as hydrocarbons, or those containing ketones and esters are also suitable for inventive PIR cross-linkers.
PIR compounds may also comprise an additional cleavable group positioned between the reporter group and the affinity tag moiety. Preferably, cleavage of this additional cleavable group can be achieved selectively with respect to cleavage of the above-described at least two labile bonds (between the reporter and the protein reactive groups). This additional cleavage site may facilitate selective removal of the affinity tag from the rest of the PIR compound or from the reporter. Examples of compounds that may be useful for this cleavage site include photo-sensitive groups comprising bonds that break upon exposure to light of a certain wavelength. Zhou et al., for example, disclose the use of photo-activatable compounds that may be used as cleavable moieties (Zhou, H., et al., Nature Biotechnology 19:512-515, 2002; incorporated herein by reference in its entirety).
Particular embodiments provide for PIR compounds having formula I:
wherein: R1 and R2 are independently H, CH3, or CH2—CH3; n=1-6;
wherein R4 is hydroxyl, or
and wherein X is selected from the following:
wherein T1-T12 are independently hydrogen, (C1-C4)-alkyl, or (C1-C4)-alkoxy; and wherein R3 is H, CH3, or CH2—CH3; wherein Q is O, N or S; wherein M is a transition metal (e.g., Zn, Fe, Co, Cu, Mn, Ni, Cr, Cd, etc);
Particular embodiments provide a compound of formula I, wherein X is:
and wherein R4 is hydroxyl.
Additional embodiments provide a compound of formula I, wherein X is:
and wherein R4 is
Further embodiments provide for PIR compounds having formula II:
wherein R1 and R2 are independently selected from hydrogen, or (C1-C4)-alkyl; wherein: n=1-6; and W and X are independently selected from:
wherein T1-T12 are independently hydrogen, (C1-C4)-alkyl, or (C1-C4)-alkoxy; wherein R3 is H, CH3, or CH2—CH3; wherein Q is O, N or S; wherein M is a transition metal (e.g., Zn, Fe, Co, Cu, Mn, Ni, Cr, Cd, etc);
wherein Y is nothing or
wherein Z is: (—CH2—CH2—O—)m; (—CH2—)q; (—CO—CH2—)r; (—CH2—CO2—)r; (—CH2—CO—CH2—CO2—)s, where m=1-5, q=5-15, r=3-7 and s=1-4 (as shown in the table below)
and wherein U is biotin, poly-histidine (6-10 residues), benzophenone (formula IV), sulfhydryl, or aryl azide (formula V).
Particular embodiments provide a compound of formula II, wherein W and X are
wherein Y is nothing; wherein Z is: (—CH2—CH2—O—)m, where m=3; and wherein U is
Additional embodiments provide a compound of formula II, wherein W and X are
wherein Y is
wherein Z is: (—CH2—CH2—O—)m, where m=3; and
wherein U is
Additional embodiments provide a compound of formula II, wherein W and X are
wherein Y is nothing; wherein Z is: (—CH2—CH2—O—)m, where m=3; and
wherein U is
Further embodiments provide a compound of formula II, wherein W and X are
wherein Y is
wherein Z is: (—CH2—CH2—O—)m, where m=3; and
wherein U is
Preferably, the compounds of formula II are of formula III:
wherein R1, R2, W, X, Y and Z are as defined above with respect to formula II.
Particular aspects provide a multivalent protein interaction reporter (PIR) compound, comprising two protein reactive moieties, each linked to a shared reporter moiety (e.g., mass reporter moiety) by a covalent labile bond that is differentially cleavable with respect to peptide bonds (e.g., by a method selected from the group consisting of collisional activation in a mass spectrometer, activation by electron capture dissociation (ECD), photoactivation and combinations thereof), wherein the reporter moiety is operatively releasable from the PIR agent upon cleavage of the labile bonds, the released reporter moiety having a characteristic identifying property or label (e.g., m/z value).
In preferred aspects, the protein interaction reporter (PIR) comprises a mass reporter moiety, and further comprises (e.g., attached to the reporter moiety) an affinity group to allow for affinity purification of the PIR compound. Preferably, the affinity group is linked to the mass reporter moiety by a selectively cleavable bone (e.g. photo-labile bond), to enable selective detachment of the affinity group from the PIR compound. In particular embodiments, the affinity group comprises a moiety selected from the group consisting of biotin, poly histidine (6-10 residues), benzophenone, sulfhydryl, aryl azide and combinations thereof. Preferably, the affinity group comprises biotin.
In particular embodiments, the protein reactive moieties comprise N-hydroxysuccinamide.
In particular embodiments, the mass reporter moiety comprises one or both of
In preferred embodiments, the protein interaction reporter (PIR) compound is of formula I or formula II disclosed herein:
Additional aspects provide a method of characterizing intermolecular or intramolecular protein interactions, comprising: obtaining a sample comprising at least one protein; contacting the at least one protein with at least one multivalent protein interaction reporter (PIR) compound, under conditions suitable for cross-linking to provide a cross-linked protein sample, wherein the PIR compound comprises at least two protein reactive moieties, each bound to a shared characteristic reporter moiety by a covalent labile bond, wherein the labile bonds can be differentially cleaved with respect to peptide bonds, wherein the reporter moiety is operatively releasable from the PIR agent upon differential cleavage of the labile bonds to provide for a characteristic released reporter moiety; differentially cleaving the labile bonds to provide for a released reporter moiety; and subjecting the cleaved products to an analysis suitable to distinguish the cleavage products by virtue of being the reporter moiety or by inclusion of a protein reactive moiety or portion thereof, wherein characterizing intermolecular or intramolecular protein interaction or structure is, at least in part, afforded.
In particular aspects, the method of further comprises, prior to differential bond cleavage, digesting the cross-linked protein sample with at least one protease.
In certain embodiments, the characteristic reporter moiety is a mass reporter moiety having a characteristic m/z value, the mass reporter moiety bound to each protein reactive moiety by a labile bond that is differentially cleavable with respect to peptide bonds by a method selected from the group consisting of collisional activation in a mass spectrometer, activation by electron capture dissociation (ECD), photoactivation and combinations thereof, and wherein subjecting the cleaved products to an analysis suitable to distinguish the cleavage products is, at least in part, by mass spectrometry.
In particular embodiments, differential cleavage of the labile bonds is by applying a first ms activation energy sufficient to provide for a released mass reporter moiety having a characteristic m/z value, and to provide for a released polypeptide or peptide linked to a protein reactive moiety or portion thereof having a characteristic m/z value. In additional aspects, the method further comprises subjecting at least one cleaved product to a second, higher ms activation energy sufficient to fragment peptide bonds.
Preferably, in such methods, the protein interaction reporter (PIR) compound is a compound of formula I or formula II as defined herein.
In yet further embodiments, the inventive methods comprise use of a plurality of distinguishable (e.g., having distinguishable protein reactive groups to provide for cross-linked products that can be independently analyzed, or otherwise analyzed) protein interaction reporter (PIR) compounds (e.g., of formula I or formula II as defined herein) to increase the sensitivity, utility and power of the methods for analysis of complex mixtures.
Inventive PIR compounds have substantial utility for the characterization of intermolecular or intramolecular protein interactions. As used herein, the term intermolecular protein interaction refers to interactions between two or more proteins or polypeptides. The term “intramolecular” protein interaction refers to interactions between different regions of an individual protein or polypeptide.
Characterization of a protein-protein interaction may occur by obtaining at least one protein and contacting the protein or proteins with a PIR agent under conditions suitable to cross-link the at least one protein with the PIR agent. The resulting mixture may contain inter, intra, and dead-end cross-linked species. Dead-end cross-linked species are cross-linking products wherein only one of the protein affinity groups are attached to a protein or polypeptide. This mixture may then be subjected to a condition, such as an activation energy, that results in cleavage of labile bonds of cleavage groups as described above, and attendant release of a characteristic reporter group.
Certain embodiments of the invention relate to conditions wherein the cross-linked species are subjected to a low-energy MS activation step that fragments the cross-linked species at the labile bonds of the PIR cleavage groups, while leaving the peptide bonds of the cross-linked proteins or peptides largely intact. In this case, the released reporter group may be detected by a characteristic m/z value by mass spectrometry. Subsequent rounds of mass spectrometry may then be used to characterize the protein regions that have been released during the first round of mass spectrometry. In such subsequent rounds, peptide bonds may be fragmented to facilitate the identification or characterization (e.g., sequencing) of the polypeptides that were cross-linked by the PIR agent.
One method of activation for fragmentation and concomitant release of a reporter group from a PIR compound is collisional activation in a mass spectrometer. In general, polypeptides are fragmented by acceleration voltages greater than 20 V. The Rink-based and indole-based cleavage groups present in PIR compounds fragment with voltages less than 20 V. Performing collisional activation at less than about 20 V generally allows for the release of intact polypeptide/peptide ions from the PIR-labeled species. Under these conditions, it is possible to obtain an accurate mass value for the polypeptide/peptides to identify them, or the polypeptide/peptides can be individually fragmented for further identification or sequencing.
In other embodiments of the invention, fragmentation at cleavage groups may be induced by photoactivation. Photoactivation may be achieved in a mass spectrometer or within the capillary used for liquid introduction. The photolabile group may be cleaved without causing peptide fragmentation. An example of a compound that may be used as a photolabile cleavage group is described by Zhou et al. (Zhou, H., et al., Nature Biotechnology 19:512-515, 2002; incorporated herein by reference).
Further embodiments of the invention provide for activation by electron capture dissociation (ECD). Low energy (1-5 eV) electrons may be captured, for example, by trapped ions in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, which is useful for peptide and protein sequence determination, because the resulting energy from charge recombination of electrons with multiply charged cations is available for backbone cleavage of peptides. ECD may be used with inventive PIRs to incorporate structures with high electron affinity that may more readily capture low energy electrons than peptides themselves. Thus, the ECD components of such PIRs will be fragmented with greater frequency than the polypeptides, leading to preferential PIR activation and peptide release.
One particularly advantageous feature demonstrated and provided herein is the capacity to distinguish dead-end and intra cross-linked peptides, and produce sequence information on peptides by combination of ECD and collisionally-activated dissociation (CAD). A useful feature of PIRs is that the mass of the reporter ion can be tuned by substituting different amino acids in the linker (e.g., spacer). The addition of an exemplary hydrophilic group and an exemplary photo-cleavable group was accomplished while maintaining the low energy fragmentation characteristics of the respective PIRs. The ability to distinguish dead-end, inter, and intra cross-linked peptides is a particularly useful feature of the instant reporter ion strategy, and provides for the effective use of the inventive cross-linkers (PIRs) in the field of interaction profiling for unambiguous identification and characterization of protein-protein interactions.
One embodiment of the present invention provides for the use of PIR compounds to cross-link a plurality of proteins present in a complex mixture, such as a cell extract or proteome. Proteins involved in protein-protein interactions vary greatly in their three-dimensional surface topography as well as in the chemical moieties present on their surfaces. To maximize the number or complexity of successful cross-linked products, a plurality of PIR compounds may be used together to characterize a variety of protein-protein interactions in a single cross-linking reaction.
Because different proteins exhibit different amino acid residues on their surface, it may be expedient to utilize multiple distinguishable PIR compounds having distinguishable protein reaction moieties; e.g., some having two or more identical protein reactive groups, and others having two or more different protein reactive groups. For example, PIR compounds having two identical protein reactive groups may be more efficient at cross-linking proteins that have similar reactive groups on their surfaces, while PIR compounds having two different protein reactive groups may facilitate the cross-linking of proteins that have different reactive groups on their surface.
Additionally, the distance between the reactive groups of proteins varies depending on the three-dimensional surface topography of the proteins forming the protein-protein complex. PIR compounds that vary in spacer arm length, for example, may promote cross-linking of proteins that vary in the distance between the amino acid chemical moieties that are reactive toward the PIR protein reactive groups.
Therefore, particular aspects provide methods comprising a plurality of different PIR compounds that vary in their protein reactive groups and/or their spacer arm lengths, wherein the PIR compounds are contacted with a complex mixture of proteins. The population of cross-linked species may be enriched or purified by the virtue of an affinity group, and subsequently analyzed, for example, by mass spectrometry using methods as described and disclosed herein.
Example 1 below shows a new mass spectrometry identifiable cross-linking strategy that has been developed and validated with an exemplary inventive PIR cross-linker (Tang, X. et al., Anal Chem 77:311-318, 2005). This cross-linker was successfully synthesized and used herein to cross-link the RNase S complex. The presence of two Rink groups and a lysine residue in the spacer chain did not hinder the cross-linking reactivity of NHS groups. Low energy MS/MS of cross-linked peptides not only readily released the distinctive reporter ions allowing rapid screening of cross-linked peptides in complex mixtures, but also preserved intact peptide chains which allowed further fragmentation by additional ms stages (e.g., MS/MS, etc.).
The cross-linker of Example 1 was synthesized based on a peptide scaffold that makes the synthesis flexible and versatile for a variety of modifications. The development of new PIR cross-linkers with specific affinity and cleavage properties will ultimately facilitate profiling of proteome-wide protein-protein interactions.
Example 2 demonstrates three exemplary PIRs and their gas phase fragmentation characteristics in low energy, collision-induced dissociation (CID) experiments in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Different fragmentation behavior of an intra-linked peptide in low energy CID and electron capture dissociation (ECD) was also demonstrated. These inventive PIRs displayed characteristic fragmentation in low energy CID experiments in FTICR-MS.
The compounds of Example 2 were constructed with acid-cleavable Rink and indole groups that have been found to fragment efficiently under low energy CAD. As shown and described herein, these groups can produce selective fragmentation of PIR labeled peptides in a low energy CID experiment in FTICR-MS. Additional features are incorporated into PIR compounds to improve the efficiency of cross-linking strategies. For example, hydrophilic groups, photo-cleavable groups, and other low energy MS/MS cleavable groups are incorporated into the inventive cross-linkers.
Example 1 A New Mass Spectrometry Identifiable Cross-Linking Strategy for Studying Protein-Protein Interactions was DevelopedChemicals. RNase S was purchased from Sigma (St. Louis, Mo.) and used without further purification. Fmoc-Rink, Fmoc-Lys (Fmoc)-OH, and HMPB-MBHA resins used for synthesis of cross-linker were purchased from Novabiochem (San Diego, Calif.). Sequencing grade modified trypsin was purchased from Promega (Madison, Wis.). Water used for preparing solution and solvent was 18 MΩ deionized water made by Barnstead Nanopure Water Systems.
Synthesis of Cross-Linker. The cross-linker PIR-0 (
Cross-Linking Reaction. RNase S was dissolved in water to make 1 mM stock solution. For cross-linking reaction, 1 mM RNase S was diluted to 10 μM in phosphate saline buffer (100 mM sodium phosphate, 150 mM NaCl, pH 7.2) and the cross-linker was added in 50-, 100-, and 150-fold molar excess over RNase S. The reaction mixture was incubated at room temperature and an aliquot was taken at 15, 30, 60, and 120 min and quenched by 1 M Tris, pH 7.5 (final concentration 40 mM).
SDS-PAGE and In-Gel Digestion. An aliquot of each reaction mixture was loaded onto SDS-PAGE for separation by using precast 4-12% gel from Invitrogen (Carlsbad, Calif.). The gels were stained with Coomassie blue (Bio-Rad, Hercules, Calif.) and imaged by Densitometer (Molecular Dynamics). The gel bands of interest were excised and destained by 50% methanol and 5% acetic acid. Performic acid oxidation method was used to dissociate di-sulfide bonds of the proteins. Performic acid was freshly made by mixing 95/5 (v/v) formic acid/30% H2O2 and incubating the mixture at room temperature for 20 min followed by incubating on ice for 5 min. 20-30 μL performic acid was used to cover the gel pieces and oxidize proteins on ice for 1 hr. The gels were then washed and dried by acetonitrile. 20 ng/μL trypsin in 50 mM ammonium bicarbonate was used for digesting proteins at 37° C. (e.g., overnight).
Nano-LC/MS/MS and Data Analysis. An electrospray-ion trap (Esquire HCT, Bruker Daltonics, Billerica, Mass.) mass spectrometer coupled with a nano-HPLC was used for nano-LC/MS/MS analysis of cross-linked RNase S complex. Nano-HPLC separation of tryptic peptides was performed with an LC packings Ultimate Nano-HPLC system equipped with a Famos™ micro autosampler and a Swichos™ micro column switching module (Dionex, Sunnyvale, Calif.). Samples were first injected by the autosampler and loaded onto a micro trap column (C18 PepMap, 300 μm×1 mm, 5 μm, LC Packings) at a flow rate of 50 μL/min with solvent A (0.1% TFA in water). The loaded samples were continuously washed with solvent A for 3 minutes to remove salts. Peptides were then eluted at a flow rate of 300 nL/min to an analytical column (C18 PepMap, 75 μm×150 mm, 3 μm, 100 Å, LC Packings) and separated using the following gradient: 0% B for 0-3 min, 20-70% B for 3-45 min, 90% B for 45-55 min, and 0% B for 55-65 min. Solvent B was 0.1% TFA in 95% acetonitrile. The eluant from the analytical column was sprayed on-line with a nano-spray emitter to the Esquire HCT mass spectrometer. The nano-spray emitter was made by applicants by etching fused silica capillary (20 μm×360 μm) with HF. The spray potential was set at 1300-1500 volts. LC/MS/MS mass spectra were acquired using HyStar™ software (version 2.3, Bruker Daltonics). MS/MS data acquisition was set in automatic mode with active exclusion based on peak intensity and a selection of exclusion peak lists. Two precursor ions were selected from each MS scan and excluded after two MS/MS scans. MS3 was performed manually by selecting specific precursor ions from MS/MS scan at specific retention time based on previous LC/MS/MS analysis of the same set of samples. For analysis of cross-linking reaction mixtures, samples were precipitated by TCA to remove the large amount of excessive unreacted cross-linker prior to loading to the nano-HPLC column. Calibration of the instrument was achieved using a solution of tuning mix (ES tuning mix, Agilent) composed of ions at m/z 322.05, 622.03, 922.01, 1521.93, and 2121.93. Data analysis and processing were performed using Bruker Daltonics Data Analysis software (version 3.1).
GPMAW™ software version 6.00 (Lighthouse Data, Odense, Denmark) was used to calculate the m/z of cross-linked peptides.
Results:Cross-Linker Design and Synthesis. Despite great enthusiasm for using cross-linking approaches, successful reports for studying protein-protein interactions on a proteome-wide scale have been scarce, due in part to the complexity of mass spectral information and the need for additional cross-linker features. Desirable goals in developing mass spectrometry identifiable cross-linking strategies using the inentive PIR cross-linkers was to simplify MS/MS spectrum of cross-linked peptides and to guide data analysis by including a specific reporter.
The conceptual components of the chemistry development for the cross-linker reagents disclosed herein are shown in
In a particular embodiment, a Rink group was coupled to a 5-mer peptide and its specific cleavage property was tested by low energy MS/MS. As expected, a majority of the fragmentation occurred at the amine position between two phenyl groups in the Rink, and the peptide chain remained intact. This fragmentation was accomplished with activation energy significantly below that needed for peptide backbone fragmentation. Therefore, two Rink groups were used in this embodiment to provide two labile bonds for the inventive PIR cross-linker. According to preferred aspects, cleavage of the two labile bonds resulted in a specific reporter molecule as shown in
The exemplary cross-linker PIR-0 was made using solid phase Fmoc peptide synthesis chemistry. Two Rink groups were treated as amino acid analogs and coupled to two the primary amines in a lysine residue. Two N-hydroxysuccinimide (NHS) esters were subsequently incorporated to make the initial cross-linker. However, the proposed modular system can readily adapt new features by using different building blocks to incorporate an affinity tag or other functional groups as shown in
According to particular aspects, the acid cleavage feature can be used for tracking cross-links by comparing mass shift prior to and following cleavage of cross-links as any other cleavable cross-linkers such as DTSSP (Bennett, K. L. et al., Protein Sci 9:1503-1518, 2000; Back, J. W. et al., Protein Sci 11:2471-2478, 2002.) This feature was not used for the studies of this Example, because the mass spectrometric cleavage features of the cross-linkers provide more benefits. As shown in
To evaluate the cross-linker and its mass spectrometric dissociation properties, the purified final product was dissolved in acetonitrile to make a 10 μM solution that was directly infused to an ESI-ion trap mass spectrometer.
Cross-Linking of RNase S Complex. Ribonuclease S(RNase S) is a complex of S-peptide 1-20 (SEQ ID NO:2) and S-protein 21-124 (redefined as 1-104; (SEQ ID NO:3)) (see
The RNase S complex is believed to be heterogeneous; (Kirchner, M. et al., 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, Tenn. 2004) other S-peptides and S-proteins were also found in the complex but only the first 15 residues of S-peptide are involved in non-covalent binding with S-protein (Wyckoff, H. W. et al., J Biol Chem 242:3749-3753, 1967). The RNase S used in this analysis was found to have S-peptides 1-20, 1-19, 1-18, 1-17, and 1-16; S-peptide 1-19 was most abundant. Nevertheless, RNase S was used as is without further purification.
Optimization of cross-linking conditions was carried out by varying molar ratios of RNase S to the cross-linker at 1:50, 1:100, and 1:150, and reaction time at 15, 30, 60, and 120 min for each reaction ratio. RNase S with no cross-linker added in the reaction buffer was used as a control. The resulting cross-linking reaction mixtures were separated by 4-12% SDS-PAGE followed by Coomassie blue staining.
As shown in the control lane of
With 100-fold excess of the cross-linker used, all upper bands appeared more intense than lower bands. As compared to a reaction ratio of 1:50, the upper bands in reaction ratio of 1:100 were located at slightly higher position indicating that some dead-end modification or intra-cross-linking of proteins may have occurred. Since 1:1 linkage of interacting proteins is most desirable, reaction ratio of 1:50 at incubation time 15 min was used for the following experiments. For in-gel trypsin digestion, the gel bands were excised carefully to include most proteins with minimum volume. The tryptic digest was further analyzed by nano-LC/MS/MS.
Analysis of Cross-Linked Proteins. The in-gel tryptic digest of cross-linked complex was further analyzed by nano-LC/MS/MS to locate the cross-links. LC/MS/MS data were acquired with the data-dependent mode. As described above, during LC/MS/MS process, detection of ions at m/z 711 indicates the potential existence of the precursor ions carrying cross-links, and observation of ions at m/z 828 is a signal that precursor ion is a dead-end modified peptide. Therefore, extracted ion chromatograms (EICs) that were built for ions at m/z 711 and 828 and the peaks in EICs provide useful clues for identifying cross-link containing peptide ions. This information can be extremely helpful since it narrows down the search window for cross-links dramatically.
As guided by these peaks, further analysis focused on these MS/MS spectra which have ions at m/z 828 and 711. Due to the specific cleavage features of the PIR, MS/MS spectra of cross-link containing peptides exhibited relatively simple fragmentation patterns, i.e., most intense peaks are generated from cleavage of labile bonds in the cross-linker.
These simple fragmentation patterns may be further utilized to differentiate dead-end, intra-, or inter-cross-linked peptides as illustrated in
As directed by the EICs of the reporter ions, an inter-cross-linked peptide was readily identified as shown in
By analyzing the MS/MS spectrum, it was further confirmed that the precursor ions were from an inter-cross-linked peptide owing to the fact that the sum of 711, neutral mass 244.9 from one peptide chain, and neutral mass 844.6 from the other peptide chain is equal to the m/z of singly-charged precursor ions. The undisrupted peptide chain at m/z 845.6 was further fragmented by another stage of MS/MS. As shown in
No other cross-links were identified except for a dead-end modification that was observed on Lys 17 of S-protein. Without being bound by mechanism, the space-filling model of the RNase S crystal structure shown in
In addition, the cross-linking reaction mixture was analyzed directly in the absence of tryptic digestion using nano-LC/MS/MS to check for modifications of S-peptide. An aliquot of cross-linking reaction mixture with a 100-fold excess of cross-linker that had an incubation time of 30 min was precipitated by TCA to remove salts and most of the unreacted cross-linker. The precipitated peptides were resuspended in 10% acetonitrile solution and loaded to the nano-LC column for LC/MS/MS analysis. Both for MS of S-peptide 1-19 and 1-20 were observed to be modified by cross-linking reactions to generate dead-end and intra-cross-linked peptides.
To get sequence information of the intact peptide chain, ions at m/z 1097.62+were isolated for MS3. As shown in
The three MS2 and MS3 spectra presented here illustrate that this cross-linking strategy can effectively distinguish dead-end, intra-, and inter-cross-linked peptides. The data also indicates that the bulky spacer chain in the disclosed cross-linker did not affect its cross-linking capability. Nevertheless, the use of the bulky spacer was able to generate a reasonable size of reporter ions to be detected by any type of tandem mass spectrometer. The release of the bulky group not only provided reporter ions, but also made it convenient for efficient fragmentation of peptide chains with short tags during MS3.
Additionally, the maximum UV absorption wavelength of the cross-linker was measured to be 280 nm, which is different than that of most peptides (214 nm). This feature may be used as an additional handle for retrieving peptides carrying inventive cross-links. It is likely that the cross-linker is not rigid in solution, and that the bonds in the cross-linker are flexible and may be folded in different confirmations to give a broad range of distances between the two reactive groups. This feature, or strategic adjustment of this feature, may have utility for general protein-protein interaction studies.
For the inter-cross-link identified between the N-terminus of S-peptide and Lys 17 of S-protein, the distance was estimated to be 14 Å from X-ray structure. In the intra-cross-linked S-peptide 1-19 (SEQ ID NO:4), the cross-link was formed between the α-amine of N-terminus and the ε-amine of Lys 1 instead of Lys 7. The maximum distance of the α-amine of N-terminus and the ε-amine of Lys 1 was calculated to be 9.1 Å. These observations further indicate that the cross-linker is fairly flexible in solution to react with the most accessible primary amines in proximity.
Example 2 New Protein Interaction Reporters having substantial utility for, e.g., Profiling Interactome by FTICR Mass Spectrometry were Developed Experimental Methods:Chemicals. Chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA) or Novabiochem (San Diego, Calif., USA) unless otherwise stated. Cross-linking reactions of Sub P(RPKPQQFFGLM) (SEQ ID NO:5) (1 μL, 1 mM) and PIR-1 and PIR-3 were performed in 100 uL PBS buffer at pH 7.2. The molar ratio of Sub P and cross-linker was maintained 1:2. The reactions were conducted for 1 h at room temperature. After 1 h, the reactions were terminated by adding 50 μL of 10 mM tris buffer. The salt was removed from the solution by SepPack™ C18 (Water, Milford, Mass., USA).
All FTICR-MS spectra were obtained with a Bruker Daltonics, 7T APEX Q-FTICR mass spectrometer by direct infusion of samples in a nano ESI source made with a fused capillary (360 μm o.d., and 20 g/m i.d) and the capillary tip was etched with 49% HF. The FTICR mass spectra were processed with the software, ICR-2LS, developed by Pacific Northwest National Laboratory (Anderson, G A, Bruce J E, Eds, ICR-2LS1995, Pacific Northwest National Laboratory: Richland, Wash., 1995). The electrospray solution was acetonitrile/0.1% TFA or 50 mM ammonium acetate unless otherwise mentioned.
Electron capture dissociation (ECD) was performed using a heated hollow cathode dispenser located outside the ICR cell to obtain the MS/MS data. The cathode dispenser was heated gradually to 1.8 A-1.9 A. Side kick trapping voltage was maintained between +6 V to −6 V. Electrons used for ECD were accelerated with 3 V. The electron injection time was 200 ms.
Synthesis of PIRs. The cross-linkers (PIR-1, PIR-2 and PIR-3), as shown in
For PIR-1 and PIR-2, the biotin and PEG group was added in the form of Fmoc-Glu (biotinyl-PEG)-OH by using standard coupling chemistry. The second lysine in the form of Fmoc-lys-ε-Fmoc was coupled to the Fmoc-Glu (biotinyl-PEG)-OH that then formed the branch point for the cross-linkers. The Rink groups, succinic acids, and N-hydroxysuccinamides (NHS) were coupled using same standard activation, coupling and deprotection chemistry.
For PIR-2 one photo-cleavable group, Fmoc-aminoethyl photo linker was introduced in between lysine and Glu(biotinyl-PEG)-OH with the same solid phase chemistry. For PIR-3 which does not contain a biotin group, one alanine residue was coupled with Fmoc-lys-ε-Fmoc to form the branch point of the linkers. The two amino functional groups of the lysine residues are then attached with (3-{[Ethyl-Fmoc-amino]-methyl}-indol-1-yl)-acetic acid. Succinic acids, N-hydroxysuccinamides were coupled as previously described (above). Reduced loading of the resin to about one third is preferred, if not necessary to get efficient coupling of the last step. The final product was cleaved using either 0.5% or 1.0% TFA in chloroform and then neutralized with pyridine. The chloroform and TFA pyridine salt was removed under vacuum until a constant weight was observed.
Formulas, molecular weights and m/e information for PIR-1, -2, and -3 are as follows:
PIR-1C83H108N12O28S
Exact Mass: 1752.7117
Mol. Wt.: 1753.8742
m/e: 1752.7117 (100.0%), 1753.7150 (92.3%), 1754.7184 (42.1%), 1755.7217 (12.6%), 1754.7159 (5.6%), 1755.7193 (5.2%), 1753.7087 (4.5%), 1754.7075 (4.4%), 1754.7121 (4.1%), 1755.7108 (4.1%), 1756.7251 (2.8%), 1756.7226 (2.4%), 1755.7154 (1.9%), 1756.7142 (1.9%), 1753.7179 (1.6%), 1754.7212 (1.5%), 1753.7159 (1.1%), 1754.7192 (1.0%)
C, 56.84; H, 6.21; N, 9.58; O, 25.54; S, 1.83
PIR-2C96H124N14O33S
Exact Mass: 2032.8176
Mol. Wt.: 2034.1508
m/e: 2033.8210 (100.0%), 2032.8176 (93.7%), 2034.8243 (52.8%), 2035.8277 (18.4%), 2035.8252 (6.6%), 2034.8218 (6.2%), 2034.8180 (5.2%), 2033.8146 (4.9%), 2036.8310 (4.8%), 2035.8168 (4.4%), 2034.8134 (4.1%), 2036.8286 (3.5%), 2035.8213 (2.7%), 2036.8201 (2.3%), 2034.8271 (1.9%), 2033.8238 (1.7%), 2034.8252 (1.3%), 2033.8218 (1.2%), 2037.8319 (1.2%)
C, 56.68; H, 6.14; N, 9.64; O, 25.96; S, 1.58
PIR-3C51H61N9O15
Exact Mass: 1039.4287
Mol. Wt.: 1040.0813
m/e: 1039.4287 (100.0%), 1040.4321 (56.7%), 1041.4354 (15.8%), 1040.4258 (3.3%), 1041.4330 (3.0%), 1042.4388 (2.9%), 1041.4291 (1.9%), 1042.4363 (1.7%)
C, 58.89; H, 5.91; N, 12.12; O, 23.07
Strategy to distinguish dead-end, intra and inter cross-linked peptides. The cross-linkers were designed with two reactive groups and two low energy MS/MS cleavable bonds. PIR-1 and PIR-2 incorporate biotin for sample enrichment. The interacting peptides are identified by looking at the reporter ion loss in the MS spectra which also can distinguish cross-linked and non specific interaction. For an intra-link peptide, the sum of the mass of the reporter ion and peptide mass should match the mass of cross-linked peptide mass (
An additional feature of protein interaction reporter-1 (PIR-1) is incorporation of a hydrophilic side chain in the cross-linker (
For studies of the gas phase fragmentation behavior of this cross-linker, protein interaction reporter 1 (PIR-1) was dissolved in acetonitrile/0.1% TFA. Water was avoided in the spray solution to prevent the hydrolysis of the reactive groups. A 10 μM solution was directly infused with a nano-electrospray source using flow rate of 20 μL/min. m/z (1753.80 Da), corresponds to the +1 charge state of calculated molecular weight of the PIR-1 (
The expected reporter ion m/z 1325.72 (+1), appeared in the MS spectra after fragmentation of two MS/MS cleavable bonds. After the cross-linking application, the m/z 1539.78, which corresponds to the cleavage of one MS/MS cleavable bond, should not appear in the mass spectrum at same m/z if coupled with a peptide. The m/z 1325.72, which corresponds to the cleavage of two MS/MS cleavable bonds, should always appear at the mass spectra at the same m/z after cross-linking, and was designated as reporter ion.
The increase of collisional trap voltage (−6 V, −9 V, −21 V) increased the intensity of one bond cleavage and reporter fragment ion intensity (
To enrich sample of cross-linked peptides, a biotin group was introduced in the PIR cross linkers. To remove the biotin tag following an enrichment step, a photo-cleavable group was introduced into PIR-2 for more efficient sample recovery (FIG. 11B). This photo-cleavable group may be cleaved by exposure to UV light at around 360 nm (Zhou, H. et al., Nat Biotechnol 20:512-515, 2002; incorporated by reference herein).
To determine if the photo-cleavable group had any effect on release of reporter ions from cross-linkers, the gas phase fragmentation of this linker was studied with FTICR-MS. The cross-linker was directly infused into the nano-electrospray source as previously described for PIR-1. This cross-linker is larger in size due to the PEG and photo-cleavable groups. In this case, the m/z 1017.52 corresponds to the intact +2 charge state of PIR-2 (
The fragmentation showed at −6 V, −9V, −12 V and −18 V effective collision energies (
In both cross-linkers PIR-1 and PIR-2, a Rink group was used to incorporate low energy MS/MS cleavable bonds. The Rink group has acid cleavable properties and is found to fragment under low energy CAD conditions in the gas phase (Rink, H.; Tetrahedron Lett. 28:3787-3790, 1987; Tang, X. et al., Anal Chem 77:311-318, 2005). In additional aspects, other acid cleavable groups can show similar low energy fragmentation features in FTICR-MS and thus have utility. For example, an additional exemplary cross-linker was designed and synthesized, and comprises an indole acetic acid group in the place of Rink groups was (
After isolation of PIR-3, the voltage settings in the hexapole collisional trap were increased as previously described (
All of the protein interaction reporters showed specific fragmentation in gas phase to release the reporter ions. The cross-linking strategy described previously, showed that sum of the m/z reporter ion fragment and original peptide mass with tags will match the molecular weight of the cross-linked peptides (
To demonstrate the utility of these cross-linkers Sub P(RPKPQQFFGLM) (SEQ ID NO:5) was coupled with PIR-1 and PIR-3. Sub P is an 11 mer peptide with molecular weight of 1347.71. The sequence contains two primary amine functional groups. One is on the lysine side chain and other is on the N-terminus. Compounds PIR-1 and PIR-3 were reacted with 1:2 molar ratio with Sub P at pH 7.2. Different molar ratios of cross-linker from 1:2 to 1:10 to 1:25 ratios were tried in the labeling step. It appeared at low 1:2 ratio highest incorporation of cross-linking (˜50%) was achieved. After purification of the sample from the salts, the dried solution was reconstituted in 50 mM ammonium acetate. The solution was directly infused in FTICR-MS with conditions as previously described above. m/z 957.86 corresponds to the +3 charge state of intra cross-linked peptides with Sub P(RPKPQQFFGLM) (SEQ ID NO:5) were isolated in quadrupole and fragmented in the hexapole collisional trap (
The voltage in hexapole collisional trap was first increased to −9V. It is clear from the spectra that two end of the Sub P was intra linked with PIR-1. Two distinctive fragment ions (m/z 1546.80 and m/z 663.37 were observed in the mass spectrum (
To demonstrate use of this application to distinguish between intra and dead-end linker, the cross-linking reaction of Sub P with PIR-3, which has indole groups substituted for Rink groups (
At a −18 V collisional trap voltage, both reporter ion (m/z 556.25 Da) and original Sub P with one tag mass (expected, 1347.71−H+128.07=1475.78 Da) m/z 1475.80 appeared in the spectrum (
To sequence the cross-linked peptide, an MS3 can be performed in the peptide and tag mass. The data presented here show that ECD spectrum of the intra-linked Sub P and PIR-1 and their low energy CID are distinctive and can be used to identify PIR labeled cross-linked peptides (
These Examples are included for purposes of illustration only, and are not intended to limit the scope of the presently claimed inventive compounds, compositions and methods.
Claims
1.-28. (canceled)
29. A method of identifying proteins participating in an intermolecular or intramolecular protein interaction within a mass spectrometry (MS) experiment comprising:
- a) obtaining a sample comprising at least one protein;
- b) contacting the at least one protein with at least one multivalent protein interaction reporter (PIR) compound, under conditions suitable for cross-linking to provide a cross-linked protein sample product, wherein the PIR compound comprises at least two protein reactive moieties, each bound to a shared characteristic reporter moiety by a labile bond, wherein the two labile bonds can be differentially relative to peptide bonds cleaved under gas phase mass spectrometer (MS) conditions with respect to peptide bonds;
- c) subjecting the cross-linked protein sample product to an MS analysis suitable to obtain a mass of the cross-linked protein sample product;
- d) differentially cleaving the two labile bonds of the cross-linked protein sample in a gas phase within a mass spectrometer to provide for cleavage products;
- e) subjecting the cleavage products to an MS analysis suitable to identify the mass of the cleavage products;
- f) analyzing the mass of the cleavage products to identify the mass of the protein participating in an intermolecular or intramolecular protein interaction by identifying the mass of the reporter moiety and which masses in the mass spectrum of the cleavage products add thereto to yield the mass of the cross-linked protein sample;
- g) determining a polypeptide sequence associated with the protein participating in an intermolecular or intramolecular protein interaction by comparing the polypeptide sequence to one or more known polypeptide or protein sequences.
30. The method of claim 29, comprising, prior to c), digesting the cross-linked protein sample with at least one protease.
31. The method of claim 29 comprising, prior to c), concentrating the cross-linked protein sample through the use of an affinity or reactive group.
32. The method of claim 31 wherein the affinity or reactive group is a biotin.
33. The method of claim 31 wherein the affinity or reactive group is a photochemical labile group.
34. The method of claim 30 further comprising concentrating the cross-linked protein sample through the use of an affinity or reactive group after digestion and before c).
35. The method of claim 34 wherein the affinity or reactive group is a biotin.
36. The method of claim 34 wherein the affinity or reactive group is a photochemical labile group.
37. The method of claim 29, wherein the differentially cleavable labile bond is cleaved by a method selected from the group consisting of collisional activation, activation by electron capture dissociation (ECD), photoactivation and combinations thereof.
38. The method of claim 29 comprising, prior to g), subjecting at least one cleavage product to a second, higher ms activation energy sufficient to fragment peptide bonds.
39. The method of claim 29 wherein the sample containing the at least one protein is a cell lysate.
40. The method of claim 29 wherein the sample containing the at least one protein is a cell.
41. The method of claim 40 wherein the unique characteristic reporter moieties have different solubility characteristics.
42. The method of claim 29 comprising, prior to g), determining the number of fragments in the mass spectrum of the cleavage products that are added to the mass of the reporter moiety to yield the mass of the cross-linked protein sample to determine if the cross-linked protein sample demonstrate an intermolecular or an intramolecular protein interaction.
43. The method of claim 42 further comprising identification of the mass of the of the reporter moiety to identify a dead-end.
44. The method of claim 29, comprises subjecting the uncleaved and cleaved products to an analysis suitable to identify the cleavage products derived from the uncleaved products by virtue of being the reporter moiety or by inclusion of a protein reactive moiety or portion thereof.
45. The method of claim 29, wherein the at least one multivalent protein interaction reporter (PIR) compound is a compound having the Formula I:
- wherein: R1 and R2 are independently H, CH3, or CH2—CH3; n=1-6;
- wherein R4 is hydroxyl, or
- wherein A and B are independently selected from:
- and wherein X is selected from the following:
- wherein T1-T12 are independently hydrogen, (C1-C4)-alkyl, or (C1-C4)-alkoxy.
46. The method of claim 29, wherein the at least one multivalent protein interaction reporter (PIR) compound is a compound having the Formula II:
- wherein R1 and R2 are independently selected from hydrogen, or (C1-C4)-alkyl;
- wherein: n=1-6;
- wherein A and B are independently selected from:
- and W and X are independently selected from:
- wherein T1-T12 are independently hydrogen, (C1-C4)-alkyl, or (C1-C4)-alkoxy;
- wherein Y is nothing or
- wherein Z is: (—CH2—CH2—O—)m; (—CH2—)q; (—CO—CH2—)r; (—CH2—CO2—)r; (—CH2—CO—CH2—CO2—),
- wherein m=1-5, q=5-15, r=3-7 and s=1-4; and wherein U is biotin, poly-histidine (6-10 residues), benzophenone, sulfhydryl, or aryl azide.
47. The method of claim 29, wherein step b comprises contacting with a plurality of distinguishable protein interaction reporter (PIR) compounds.
48. The method of claim 47, wherein at least one of the distinguishable protein interaction reporter (PIR) compounds is of formula I of claim 45.
49. The method of claim 47, wherein at least one of the distinguishable protein interaction reporter (PIR) compounds is of formula II of claim 46.
50. The method of claim 29, wherein the labile bonds, in each case, comprise a carbon-nitrogen bond that can be differentially cleaved with respect to peptide bonds.
51. The method of claim 50, wherein the carbon atom of the differentially cleavable carbon-nitrogen bond, in each case, is adjacent to a moiety selected from the moiety group consisting of aromatic moieties, N-functionalized heterocyclic aromatic moieties, substituted biphenyl compounds, dithiols, phosphates, metal ligand complexes, and other moieties with significant π-delocalization.
52. The method of claim 51, wherein the N-functionalized heterocyclic aromatic moiety is selected from the group consisting of substituted or unsubstituted indole moieties, pyridyl-based moieties, and combinations thereof.
53. The method of claim 50, wherein the nitrogen atom of the differentially cleavable carbon-nitrogen bond, in each case, comprises a secondary or tertiary amine moiety.
Type: Application
Filed: Apr 14, 2009
Publication Date: Feb 25, 2010
Applicant: Washington State University (Pullman, WA)
Inventors: James E. Bruce (Seattle, WA), Xiaoting Tang (Seattle, WA), Gerhard Munske (Pullman, WA)
Application Number: 12/423,459
International Classification: G01N 33/53 (20060101);
