One tool to study protein-protein interactions in a physiological context is affinity enrichment of the protein of interest followed by detection of its binding partners using either immunodetection methods or mass spectrometry [ 3 ].
However, this classical immunoprecipitation method has two drawbacks. Weak interactions could be missed, if stringent wash conditions are applied. In contrast, nonstringent conditions may enable the identification of more proteins, but many of these could be false positives only binding the bait protein during sample preparation.
One approach to solve this problem is applying covalent cross-linking to intact cells and thereby stabilizing protein-protein interactions, including very weak and transient ones [ 3 ].
After this fixation step, highly stringent conditions can be used during cell lysis and affinity enrichment, minimizing the risk of identifying false positives. Several cross-linkers varying in spacer arm lengths, reaction groups, and other properties are commercially available. One of the shortest available cross-linkers is formaldehyde 2.
The experimental conditions used in these applications lead to a very tight network of cross-links, which prevents the precipitation of one protein of interest as required for protein-protein interaction studies. However, lower formaldehyde concentrations 0. The application of formaldehyde as a cross-linker has several advantages. Only closely associated proteins can be cross-linked due to the small size of formaldehyde. Furthermore, its high permeability towards cell membranes enables cross-linking in the intact cell, without addition of organic solvents such as dimethyl sulfoxide as necessary for other cross-linkers.
Formaldehyde is also thought to allow very fast cross-linking and the stabilization of transient interactions [ 4 ]. Finally, formaldehyde is available in almost every laboratory at costs that amount to only a fraction of other cross-linkers.
However, formaldehyde cross-linking is not yet an established standard method and many questions regarding the optimal experimental conditions and the usability of antibodies for pull-down of proteins after formaldehyde treatment remain.
For example, epitopes recognized by antibodies raised against endogenous proteins could be destroyed by formaldehyde modification, which would prevent their application [ 9 ]. Similarly, the physiological environment of a protein of interest, and the type and extent of its interactions may also affect the experimental outcome. Therefore, we decided to investigate different aspects of formaldehyde cross-linking in more detail using the transmembrane protein integrin as a model.
Integrins are membrane spanning heterodimeric complexes that play important roles in cell adhesion and migration processes by interacting with components of the extracellular matrix [ 10 ]. The biggest subgroup with 12 members is formed by containing heterodimers [ 11 ].
Before being able to bind a ligand, integrins have to be activated through an intracellular process termed inside-out signalling. Activated talin then binds to the intracellular tail of the integrin and causes conformational change of the two integrin chains.
This allows binding of extracellular ligands, which drives the cytoplasmic tail of the integrin to bind additional adaptor proteins, establishes a connection to the cytoskeleton and leads to the delivery of the external signal outside-in signalling.
Several intracellular interaction partners have been described for integrins, despite the shortness of the intracellular tail of integrins, which varies between 40 and 60 amino acids.
However, these interactions cannot take place simultaneously, but depend on the activation status of the cell and the integrin. The detailed binding procedures as well as the signalling processes triggered by these are not fully understood.
For example, talin and kindlin both interact with integrin and a crosstalk between them is assumed. However, it remains unclear, whether both proteins connect with the integrin at the same time or binding occurs sequentially [ 14 ].
Studying the interaction partners of integrins using the formaldehyde cross-linking approach, which should be able to identify transient and indirect interaction partners of proteins, may shed more light on these processes and would therefore be very valuable. In the present study, we report the optimization of a protocol applying formaldehyde cross-linking combined with immunoprecipitation and mass spectrometry Figure 1 a to analyze the interaction network of integrin.
Human platelets were isolated from healthy human volunteers as described earlier [ 15 ]. This was approved by the University of British Columbia Research Ethics Board and informed consent was granted by the donors.
Briefly, whole blood was drawn from the antecubital vein into 0. Platelets were isolated by centrifugation and washed in physiological buffer. All anti-integrin antibodies were monoclonal mouse anti-human antibodies provided by John Wilkins Manitoba. Formaldehyde solution was obtained by dissolving 0.
The solution was filtered 0. The supernatant was removed and the reaction was quenched with 0. Cells were transferred to a smaller tube, spun, washed once in 1. After 30 minutes, cell lysates were treated with 50 strokes using a Dounce homogenizer. Control cells were treated exactly the same way, except that they were resuspended in PBS instead of formaldehyde solution.
Protein concentration was determined using a BCA assay Pierce. All steps were performed with mild agitation at C. For mass spectrometric analysis, lysates were precleared by incubation with the same amount of beads for 2 h before the antibody was added.
The supernatants of the immunoprecipitations were kept for analysis and the beads were washed either twice with PBS for western blot analysis or three times with RIPA buffer for mass spectrometric analysis.
Quantitation of immunoblot analysis was performed using the software ImageJ [ 20 ]. Silver staining was performed using a modified protocol of Gharahdaghi et al. Gels were incubated in 0. The reaction was stopped by addition of acetic acid. Bands of interest in silver-stained gels were excised and destained as described [ 21 ].
In-gel digestion was performed using standard procedures [ 22 ]. Briefly, samples were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin Promega in ammonium bicarbonate overnight.
Maisch GmbH, Germany with water:acetonitrile:formic acid as the mobile phase with gradient elution. Known contaminants such as keratin were removed from the protein lists.
The usage of formaldehyde as cross-linking reagent has to be evaluated in order to determine the optimal balance between highest yield of complex formation and lowest artefact generation [ 4 ]. Optimization has to be performed for each protein of interest, as it is dependent on the physiological environment of the protein itself and can vary for example between cytosolic and membrane proteins.
Three main parameters play a critical role during formaldehyde cross-linking: the reaction temperature, the incubation time and the formaldehyde concentration.
The temperature dependency had been studied in our laboratory earlier and a difference between incubation at C and C could not be detected unpublished results. Room temperature is advantageous, as it results in the most convenient and easiest approach possible. In addition, we chose not to increase the incubation time to more than 10 minutes, as the advantage of using formaldehyde as a cross-linker is the short reaction time it requires, which minimizes the formation of unspecific cross-links and allows the fixation of transient interactions.
Moreover, model studies on peptides had shown that incubation time and formaldehyde concentration are complementary [ 24 ]. Therefore, we decided to limit our study to the usage of different concentrations of formaldehyde in terms of our model protein integrin complex. Jurkat cells were chosen for studying integrin interactions, as this human cell line expresses high amounts of this integrin and has been used extensively for its investigation before [ 25 ].
Different concentrations of formaldehyde 0. Cells were lysed under stringent conditions using RIPA buffer to destroy weak and noncovalent interactions, and protein amounts were determined. Lysates of formaldehyde treated cells contained lower amounts of protein than nontreated cells.
This can be explained by the formation of insoluble complexes, for example, nuclear proteins being cross-linked to DNA, which were precipitated during lysis and removed in the insoluble pellet. This effect was visible during sample generation: lysis of nontreated cells using the stringent RIPA buffer led to the release of DNA, which formed a cloudy precipitate and could be easily removed. In contrast, a cloudy suspension was generated during lysis of formaldehyde treated cells and the pellet had a different consistency, which required a longer centrifugation period to become separated.
Consistent with this observation, nuclear proteins were not detected in the lysate by immunoblot analysis. However, membrane proteins were overrepresented due the loss of nuclear proteins data not shown.
We recommend using this difference in appearance as an early indication of successful cross-linking. Optimization of the formaldehyde cross-linking protocol involving integrin required a read-out of the cross-linking efficiency, for example, the detection of a complex containing the integrin.
Formaldehyde cross-links are reversible during the standard sample preparation for SDS PAGE analysis Figure 1 b , which includes boiling in reducing Laemmli buffer [ 19 ], thus cross-linked complexes would not be detected under these conditions [ 8 ].
However, by reducing the incubation temperature to C, cross-linked complexes are not fully destroyed and remain detectable Figure 1 c [ 5 ]. Membrane protein studies by gel electrophoresis are often performed at even lower temperatures 37—4 C. Initial immunoprecipitation experiments we had performed indicated that at C the antibodies used for pull-downs would not dissociate into their heavy and light chains data not shown.
Instead, during gel electrophoresis the intact antibodies would migrate at molecular weights that would overlap with the cross-linked complexes and therefore interfere with their detection. Consequently, we decided not to use temperatures lower than C in our experiments. We could recognize a higher molecular weight complex containing integrin in samples treated for 5 min at C, whereas this band was nearly undetectable after boiling for 10 minutes at C Figure 1 d. Therefore, we concluded that we were visualizing a cross-linked complex containing integrin.
However, integrin was also found in the monomeric form at appr. This could be due to incomplete cross-linking, as the conditions applied during formaldehyde cross-linking do not lead to a high extent of protein cross-linking, leaving a large fraction of integrin noncross-linked.
Alternatively, incubation at C may lead to partial reversal of formaldehyde cross-links and release of integrin even at a lower temperature.
Equal amount of lysates of the samples generated using different concentrations of formaldehyde were analyzed by immunoblotting Figure 2 a. No complex was detected in the control, in which cells were treated under cross-linking conditions but without formaldehyde. With 0. The amount increased slightly at higher concentrations of formaldehyde, but then settled, with no apparent difference between 1. These were plotted together with the total protein concentrations of the lysates to determine the optimal cross-linking parameters Figure 2 b.
The amount of monomeric integrin did not vary significantly between the different formaldehyde concentrations, even though the amount of the complex was increasing Figure 2 b. This apparent contradiction can be explained by the aforementioned observation that membrane proteins are enriched during formaldehyde treatment relative to other cellular components.
Thus, by loading equal total protein amounts in each lane, increasingly higher amounts of total integrin were applied. Unfortunately, this variation cannot be compensated using loading controls, as the exact amount of each cross-linked protein cannot be predicted.
However, increasing the extent of cross-linking may also result in the formation of larger complexes by involving proteins that do not directly bind to integrin , including the cytoskeleton. This would lead to the formation of more extensive, heterogeneous cross-linked complexes that would provide a larger surface to which nonspecific proteins can bind during sample processing.
As a result, an increasing amount of abundant cytoskeletal proteins and common contaminants would be identified that would not be considered specific interactors.
Therefore, to minimize such apparent artefact generation, we decided to perform the following investigations using more stringent conditions by applying 0. This increases the likelihood of missed identifications of specific interactors of low abundance and of interactions of low stoichiometry.
Even though higher formaldehyde concentrations would counter this effect, they would also result in more artefacts, hence differential analysis using multiple formaldehyde concentrations would still not be able to distinguish between specific interactors and artefacts. Instead, individual follow-up experiments for each identified protein would be required to determine its specificity. As the focus of this study was not to obtain an extensive list of putative interactors, but rather to demonstrate the general validity of the approach, we chose lower formaldehyde concentrations and high stringency.
Users interested in maximizing the number of captured proteins should consider using higher formaldehyde concentrations instead.
Studying interaction partners using formaldehyde cross-linking and affinity purification requires the antibody to still recognize its target after reaction with formaldehyde.
In our earlier studies utilizing myc-tagged proteins, the antibody 9E10 was shown to be suitable for formaldehyde treatment, as it still precipitated protein complexes [ 5 ]. The use of a tagged, exogenous version of integrin would result in additional risks coming with this approach, such as identification of false positives binding to the tag, altered protein localization, or general changes in the protein environment due to elevated expression levels.
Antibodies recognizing the endogenous protein are required to exclude these influences. Not all monoclonal antibodies are expected to be suitable in this technique, as some epitopes might be destroyed by formaldehyde modification of amino acids Figure 1 a. We wondered whether it is possible to predict the applicability of an antibody in the formaldehyde cross-linking approach. Therefore, we analyzed eight different and mostly well studied monoclonal anti-integrin antibodies Table 1 and Figure 3.
First we wanted to know whether these antibodies were able to immunoprecipitate integrin from cell lysates generated using the lysis conditions we apply during cross-linking. These two bands likely represent two differently -glycosylated integrin chains that have been detected on the cell surface of several cell types [ 27 ].
In contrast to our results, Meng et al could not detect the low-mass integrin in Jurkat cells, but they only investigated the cell membrane whereas we precipitated integrin from whole cell lysates. This suggests that the low-mass form may be retained in the endoplasmatic reticulum. A closer look at the known properties of the anti-integrin antibodies revealed that five of them had been shown to stimulate integrins when added to living cells, while two showed inhibitory character Table 1.
However, as we were planning to use these antibodies for precipitation of integrins from cross-linked and thereby fixed cells, these properties should not affect our experiments. More importantly, the epitopes of some of these antibodies are known and located at very different positions in the extracellular part of the protein: N29 and JB1A detect continuous peptide sequences close to the protein -terminus Figure 3 b.
In contrast, B3B11 and JB1B detect continuous sequences at the opposite end of the extracellular part of integrin Figure 3 b , where also the discontinuous epitope of 2C18 is found [ 29 ].
B44 is known to recognize a different part of the protein than the other antibodies but the precise epitope is unknown. Since the B44 antibody is working in immunoblotting experiments, its epitope is expected to be composed of a continuous sequence [ 26 ].
In contrast, 3S3 cannot be used for immunoblotting indicating a discontinuous epitope. We further modelled the integrin structure on integrin to gain insight into the three-dimensional location of the known epitopes Figure 3 c. As expected the epitopes are located in very different parts of the protein.
Additionally it is known that the epitopes of N29 and B44 are hidden in the inactivated form of integrin as on some cell lines they can only detect the receptor after it had become activated [ 29 ]. To assess whether the antibodies would also be able to work after formaldehyde treatment, we had a closer look at the epitope sequences, which are known for four of them Figure 3 b. It had been shown that the amino acids, which are formaldehyde-modified first during the short incubation times used under cross-linking conditions, are lysine, cysteine and tryptophan [ 24 ].
Our next step was to determine whether the antibodies found to precipitate integrin in the previous section can detect and pull down the integrin complex.
Jurkat cells were treated with or without 0. Cells were lysed and the lysates were used for an immunoprecipitation using the antibody JB1B and a control pull-down which was performed without an antibody Figure 4 a.
The higher molecular weight complex containing integrin was only detected in formaldehyde treated cells and was only precipitated when the antibody was added. No complex was detected in the supernatant of the immunoprecipitation, indicating its complete pull-down.
Furthermore, no loss in total integrin precipitation was observed, indicating that formaldehyde treatment did not destroy the JB1B epitope. This had been expected, as the epitope does not contain many of the easily modifiable amino acids. As a next step, all of the other antibodies were used to precipitate integrin from formaldehyde treated and non-treated Jurkat cells Figure 4 b.
All of them were able to precipitate the target protein even after formaldehyde treatment, indicating that none of their epitopes was destroyed in such a way that it would prevent precipitation. Even for N29 and JB1A, whose epitopes contain a high percentage of easily modifiable amino acids, no fundamental loss in integrin precipitation was observed.
However, not all antibodies were equally able to precipitate integrin containing complexes: B3B11, JB1A and 3S3 precipitated the complex completely, since no integrin was detected in the high molecular range of the supernatant.
Only partial precipitation of a high molecular weight complex was observed using the antibodies 2C18 and 6S6, where part of the complex was detected in the supernatant of the precipitation. No precipitated complex was detected using the antibodies N29 and B As these antibodies were able to precipitate integrin of formaldehyde treated cells, it is unlikely that their epitopes were destroyed during the process.
However, as mentioned earlier N29 and B44 have both been shown to detect activated integrin on cells, whereas their epitopes are disguised on non-activated cells [ 29 ]. Stringent lysis of cells destroys this conformational limitation and allows detection by the antibodies. In contrast, cross-linking preserves the conformation and therefore a non-activated complex would be undetectable and not precipitated by these two antibodies. On the other hand, they could be able to precipitate an activated complex which in turn would not be recognized by some of the other monoclonal antibodies.
This limitation of antibody usage does not only apply to formaldehyde cross-linking, but restricts every approach where protein complexes are precipitated by antibodies, including the traditional coimmunoprecipitation method which uses gentle lysis conditions to preserve protein interactions. Finally we asked whether a higher degree of modification induced by higher concentrations of formaldehyde would impede the precipitation of integrin using the antibody JB1B.
Therefore we tested the ability of JB1B to pull down complexes from cell lysates, which were obtained after treatment of Jurkat cells with varying formaldehyde concentrations. JB1B was able to precipitate integrin and the complex regardless of the formaldehyde concentrations applied, which was shown by analysis of the pull-downs as well as the supernatants of the immunoprecipitations Figure 4 c : no protein was detected in the supernatants, whereas integrin was detected in the range of the monomeric protein and the high molecular weight complex in the immunoprecipitations.
We concluded that JB1B is the most universal of the anti-integrin antibodies we had tested, and therefore the most useful for formaldehyde cross-linking. Western blot analysis had shown that we precipitate a high molecular weight complex containing integrin after formaldehyde treatment. As the other components of this complex were unknown to us, we used mass spectrometry to identify them. Furthermore we consistently identified integrin 4. The receptor formed by these integrins is VLA-4 very late antigen 4 , a known receptor found on T cells [ 31 ] and a high abundant integrin on Jurkat cells [ 25 ].
Two of these experiments also allowed the identification of additional proteins Table 2. The listed proteins were not detected in control experiments using either non-formaldehyde treated cells or immunoprecipitations performed without the antibody when analysing gel bands at masses corresponding to the integrin containing complex.
Integrin 5 had been demonstrated to be expressed together with integrin on Jurkat cells [ 25 ], whereas integrin is known to form a collagen-recognizing receptor with integrin on endothelial cells [ 32 ]. Even though only few peptides of each protein were sequenced, the identification of the integrin subunits is reliable as they do not share high homologues sequences and all sequenced peptides were unique for each integrin.
The other identified proteins are not known to interact directly with integrin [ 13 ]. As a member of the cytoskeleton, actin is intertwined with integrin function and associated with integrins through adaptor proteins such as talin [ 10 ].
However, an adaptor was not consistently identified in these immunoprecipitations. Furthermore actin is a highly abundant protein and its identification has to be judged carefully. Thus we cannot exclude that these proteins are non-specific background, even though they were not detected in the control samples. As formaldehyde cross-linking could also occur with abundant proteins that colocalize but have lower binding affinity to integrin , this may explain the presence of abundant proteins.
Alternatively, proteins that do not colocalize but show medium to high affinity may form a non-cross-linked complex in the lysate that is sufficiently stable not to be destroyed by the incubation temperature of C that is applied prior to gel separation. This scenario may also be true for most of the other proteins Desmoglein-1, Filaggrin-2, Junction plakoglobin and C1orf68 , which are highly expressed in skin cells.
These could be contaminants similar to keratins which were removed before data interpretation occurring during sample preparation. The last identified protein arginase-1 is found in the liver and plays an important role in arginine metabolism [ 33 ], a connection to integrin is not known.
On the other hand, formaldehyde cross-linking would also stabilize interactions between two proteins that do not directly interact but have a common binding partner. Therefore, such unexpected findings may still provide valuable information.
No known adaptor proteins like talin or tensin were consistently detected in the complex. This indicates that the precipitated complex is mostly in an inactive state, where the two integrin chains form a stable and very compact complex and no adaptor proteins are bound to the intracellular tail.
This matches our earlier observation that N29 and B44, which were shown to be unable to detect inactive integrin , did not precipitate this complex. All our results imply that most of the integrin found on the Jurkat cells is not activated, which is in contrast to an earlier study where Jurkat cells have been shown to express constitutively active integrin [ 25 ]. However, it has to be considered that cell lines change their behaviour over time, thus it might be that the Jurkat cells used by them and by us differ in this aspect.
In addition to our studies using Jurkat cells, we wanted to apply our approach also to a different and physiologically more relevant system, and therefore used human platelets. Platelets express high amounts of integrins, which play an important role in platelet aggregation. Even though integrin is not the most abundant integrin found on platelets, it forms three different receptors , , and is involved in platelet adhesion [ 34 ]. Human platelets were treated with and without formaldehyde and the antibody JB1B was used to precipitate integrin from the lysates.
Since the cell number is the same, I'm therefore drastically increasing the CH2O:cell ratio even if the concentration is technically the same. Concentrations don't crosslink; molecules do. In any case, a crosslinking concentration assay never hurt anyone!
Now if you have less cells but much more volume of Formaldehyde, I can see how its not sufficient to quench everything. How much volume for how many cells if I can ask? Location: San Antonio. This is not the first time I have seen shearing like this using the Covaris.
I think the glycine thing is a "red herring" here and I would suspect your shearing methodology is the problem. Have you tried any other sonicators?
I would hope so after two years. Last edited by theduke; at PM. Originally Posted by theduke. I don't think there is much doubt that glycine quenches formaldehyde. I would drop that line of thinking quickly if I was you. There is too much literature and too much evidence against you and it can't all be wrong.
There is also no doubt that fixing time per se influences shearing efficiency. Something else is going on with your experiments. It looks to me that you are massively over-crosslinking. This is where I think your problems are.
Something doesn't smell right. Are you doing the quenching etc in suspension? That is correct. Since CH2O has a molecular weight of about 30, it follows that there are 30 g per mol. How can something that reacts to stop formaldehyde from crosslinking further possibly work at a mM to mM ratio? Again, I added mM glycine FIRST before formaldehyde and the cells went right on to over-crosslink all of the material was crosslinked.
If I'm crosslinking in media, there should be a bit of extra quenching power from its components. Despite this, the amount of glycine I'm adding doesn't actually quench. If quenching doesn't actually happen, that means my cells are crosslinking while I aliquot them into 50 ml tubes another minutes and while they pellet another 5 min.
Instead of crosslinking for 30 seconds, I'm really crosslinking for 30 minutes, hence the reason my sonication sucks. I can't drop the line of thinking that glycine isn't quenching formaldehyde in these conditions because I have reproducible, empirical evidence showing it doesn't and it explains my sonication failures despite two years of optimization using protocols directly from the labs of Richard Young and the ENCODE Consortium.
That's why I think that there are other factors at play, such as the number of molecules not the concentration per cell. Originally Posted by kylini. Well, I don't know what career-stage you are at, but spending two years figuring out sonication and ChIP-Seq is far, far, far too long in my opinion.
Fun update! Here's the edited experimental setup: Here's the result: In conclusion, glycine does not quench formaldehyde but Tris does. If you have quenching issues, give Tris a try! It's certainly been used in the literature; this just confirms that it works. Location: Woodbridge CT. Isn't there serum in the cell culture media that you're adding formaldehyde to? Originally Posted by Joann. Originally Posted by austinso. All times are GMT
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