This article focuses on using crosslinkers to analyze protein–protein interactions. For a fuller discussion of crosslinkers and protein crosslinking, see Overview of Crosslinking and Protein Modification and the other articles referenced therein.
Crosslinking reagents covalently link together interacting proteins, domains or peptides by forming chemical bonds between specific amino acid functional groups on two or more biomolecules that occur in close proximity because of their interaction. Commercially available crosslinking reagents have a wide range of characteristics, including:
Chemical structure of DSS.
Chemical structure of SMCC.
Chemical structure of BS3.
Chemical structure of DSP.
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Continue reading: Overview of Protein-Protein Interaction Analysis
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Besides the transient and sometimes tentative nature of some protein–protein interactions, the formation of these complexes can change in response to any number of stimuli, including changes in pH, temperature and osmolarity, and either the lack of a specific protein or co-factor or the introduction of a protein with which the protein(s) do not normally interact.
The benefit of in vivo crosslinking is that the protein–protein interaction can be captured in its native environment, which limits the risk of false positive interactions or the loss of complex stability during cell lysis. For in vivo crosslinking, hydrophobic, lipid-soluble crosslinkers are expected to be used if the target protein is within or across cell membranes, while hydrophilic, water-soluble crosslinkers can be used to crosslink cell surface proteins, such as receptor–ligand complexes. This representative data provides an example of various reagents used for in vivo crosslinking.
Comparison of several in vivo crosslinking methods. HeLa cells treated with 1% Formaldehyde (HCHO) or 1 mM homobifunctional NHS-ester crosslinker (Thermo Scientific DSG and DSS) in PBS for 10 minutes before quenching. A fourth set of HeLa cells were treated and crosslinked for 10 minutes with 4 mM Photo-Leucine, 2 mM Photo-Methionine (Photo-AA) according to the procedure. Formaldehyde-treated and NHS-ester–treated cells were quenched with 100 mM glycine (pH 3) and 500 mM Tris (pH 8.0), respectively for an additional 15 minutes. One million cells from each condition were then lysed and 10 µg of each sample was heated at 65°C for 10 minutes in reducing sample buffer containing 50 mM DTT followed by analysis by SDS-PAGE and western blotting with Stat3 specific antibodies (Cell Signaling). GAPDH (Santa Cruz) and beta-actin (US Biologicals) were blotted as loading controls.
Due to the high concentration of proteins in cells, crosslinkers with shorter spacer arms are usually recommended for in vivo crosslinking approaches to increase the specificity of conjugating actual interacting proteins as opposed to proteins that just happen to be in close proximity to each other during incubation with the crosslinker.
Although in vivo crosslinking can yield physiologically relevant, stably-crosslinked complexes for analysis, optimizing this approach can be difficult, as the reaction conditions cannot be tightly controlled and crosslinkers react with a wide array of proteins that all present functional groups against which crosslinkers specifically react.
In vitro crosslinking can better target specific crosslinking events, because more reaction conditions can be tightly controlled, including the pH, temperature, concentration of reactants and purity of the target protein(s). The ability to control all aspects of a conjugation experiment results in better analysis due to greater resolution of protein–protein interactions. Additionally, in vitro methods of conjugation allow researchers to modify interacting proteins, such as adding polyethylene glycol groups (PEGylation), blocking sulfhydryls or converting amines to sulfhydryls. Also, a greater variety of crosslinking reagents, both hydrophobic and hydrophilic, are available for in vitro applications. This representative data was produced using the amine-reactive crosslinking reagents, DSS, BS3, and DSSO.
Comparison of BSA crosslinking efficiency by SDS-PAGE. Different crosslinkers were incubated with BSA at molar excess of crosslinker to protein (e.g., 20-, 100- or 500-fold). Crosslinking efficiency is shown by decreased mobility by SDS-PAGE and varied by crosslinker type, solubility and concentration.
Obviously, the disadvantage of using in vitro methods to conjugate proteins is the lack of physiological conditions. Additionally, rupturing and solubilizing membranes can disrupt protein–protein and protein–membrane interactions.
Because a myriad of crosslinking reagents are commercially available for many different applications, the key determinant in deciding to use in vivo or in vitro crosslinking is the target protein, specifically in term of its:
Correct identification of protein-protein interactions first requires the selection of the best crosslinker to use. Because there are multiple amino acid functional groups that may react with different crosslinkers, an empirical strategy of screening multiple types of crosslinkers should first be performed to identify the target protein conjugate. The crosslinkers tested may vary in:
Once the target interaction is detected by any of the methods listed below, then the protocol can be fine-tuned to optimize detection by adjusting crosslinker concentration, pH and other reaction conditions.
The starting protein concentration or number of cells should be empirically determined for in vitro and in vivo crosslinking protocols, respectively. For in vitro crosslinking, the protein solution should be prepared in a nonreactive buffer, such as phosphate-buffered saline (PBS), which has the proper pH for the specific crosslinker. For in vivo crosslinking applications, cells should be in the exponential phase of growth and at a subconfluent density during the crosslinking procedure. To avoid the possibility of culture media reacting with the crosslinker, the media can be replaced with PBS through a series of cell washes.
Crosslinkers should be prepared as per the manufacturer's instructions; hydrophobic crosslinkers are first dissolved in the appropriate solvent, such as methanol or acetone. The optimum amount of reagent to add also depends on the crosslinker, but usually a 20- to 500-fold molar excess (relative to the lysate protein concentration) is appropriate. Ensure that pH of the reaction buffer is favorable for the crosslinker. Most amine-reactive crosslinkers require alkaline pH for activity.
The crosslinking reaction time may also be important, depending upon the experiment and crosslinker being used. While 30 minutes is a good incubation time to start with, multiple experiments can be performed concurrently to test other lengths of time to determine the optimal time of incubation with the specific crosslinker. Long incubation periods should generally be avoided, not only because it may cause formation of large, crosslinked protein aggregates, but also because the crosslinker may lose stability. In cases where extended incubation periods are required, though, fresh crosslinker can be added at specific time points throughout the procedure to maintain the proper molar ratio of reagent and maximize the formation of the target product. The formation of aggregates due to extensive crosslinking, though, should also be considered in determining the optimal reaction time.
With most amine-reactive crosslinkers used for protein–protein interaction analysis, the reaction can be halted at the desired time by adding excess nucleophile, such as Tris or glycine, which out-competes the lysate proteins for reaction with the crosslinker. The crosslinked product can then be purified through multiple approaches, including precipitation, chromatography, dialysis or ultrafiltration.
A rapid method that combines quenching the reaction and denaturing the proteins in preparation for gel electrophoresis is to add sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) buffer, which contains both Tris and 2-Mercatpoethanol, and then boil the solution for 5 minutes. The sample can then be directly analyzed by gel electrophoresis.
Continue reading: Overview of Crosslinking and Protein Modification
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Crosslinking is typically used to capture and stabilize transient or labile interactions so that they can be further isolated and analyzed by downstream methods such as electrophoresis, staining, western blot, immunoprecipitation or co-immunoprecipitation and mass spectrometry.
When two proteins are covalently crosslinked, the gel migration patterns of both proteins shift in relation to the uncrosslinked proteins. Therefore, if antibodies that detect each target protein are available, the most straightforward method to detect the shift of the interacting proteins is by SDS-PAGE and western blot analysis.
Both immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are methods to detect protein expression and protein–protein interactions, respectively, via affinity purification. Crosslinking is commonly performed in both applications, either alone or in combination with affinity binding, to immobilize antibody to the beaded support and or freeze weak antibody–antigen interactions to prevent sample loss during immune complex extraction. Crosslinking is also used to stabilize transient or weak protein–protein interactions prior to co-IP protocols. Following both approaches, samples are commonly analyzed by SDS-PAGE.
Co-immunoprecipitation of cyclin B and Cdk1. The Thermo Scientific Pierce Protein A/G Magnetic Beads bind to Cdk1 antibody complexed with Cdk1. Cyclin B is bound to the Cdk1, and is captured along with its binding partner.
When analysis by mass spectrometry (MS) is available, the peptide fragments that are crosslinked between interacting proteins can be identified by the change in mass resulting from the attached crosslinker molecule. In this approach, identical samples are crosslinked with either deuterated (heavy) or nondeuterated (light) crosslinkers. The crosslinked proteins are then pooled together and analyzed by MS to identify and quantify the heavy product based on its shift from the light product. This method also commonly employs SDS-PAGE as a first-stage purification step prior to digestion in preparation for MS analysis.
BSA crosslinked peptide spectra. BSA crosslinked peptide spectra were identified by MS2-MS3 method and XLinkX using DSSO crosslinker. XlinkX software uses unique fragment ion patterns of MS-cleavable crosslinkers (purple annotation) to detect and filter crosslinked peptides for a crosslinked database search.
Learn more about how to desalt, buffer exchange, concentrate, and/or remove contaminants from protein samples, immunoprecipitation and other protein purification and clean up methods using various Thermo Scientific protein biology tools in this 32-page handbook.
Continue reading: Overview of Western Blotting
Continue reading: Overview of the Immunoprecipitation (IP) Technique
Continue reading: Co-immunoprecipitation (Co-IP)
Continue reading: Overview of Mass Spectrometry for Protein Analysis
Explore: Western Blotting
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