This work was funded by the Wellcome Trust (WT098051).

<ol>
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The authors have nothing to disclose.

Here, we describe the use of affinity purification followed by blue native gel electrophoresis in combination with quantitative mass spectrometry to resolve protein complexes. This approach offers a method to unravel one-dimensional protein interaction lists into functional protein assemblies.
We demonstrate the method based on the use of epitope-tagged proteins. However, if a cell line expressing a tagged protein is not available, an alternative might be to use antibodies against the protein of interest, provided there is a peptide available to achieve native competitive elution. The amount of starting material and quantity of beads might need to be modified depending on the expression level of the target protein. We typically perform this protocol with 2-5 x 108 cells starting material, and this is sufficient even for proteins with low expression level. The lysis buffer composition should be chosen empirically to achieve near to complete solubilization of the bait. This might be challenging for some types of proteins, in particular chromatin binding or membrane proteins. Alternative options include increasing the amount of salt, provided that the protein complex under study is stable in high salt, or using sonication and/or nuclease treatment for chromatin binding proteins 20,29. In the case of membrane proteins, switching detergent to DDM or digitonin may be advisable 30. In the case of DNA binding proteins, it is useful to include a nuclease such as benzonase during the purification step 20. Complete removal of nucleic acids ensures that the interactions detected occur between proteins and are not mediated by DNA.
A critical element to consider when using this approach is the stability of the complex under investigation. The procedure is long and may involve preserving the protein complex overnight. We have achieved good success with two chromatin remodeling complexes (D. Bode and M. Pardo, data not shown), but this should be evaluated. The affinity purification step may be shortened if required.
Alternative techniques that allow native fractionation, such as size exclusion chromatography, have been widely used for over 50 years to characterize protein complexes. We and others have shown that the resolution of blue native PAGE is superior to that achieved using size exclusion chromatography 20,31,32,33. Another advantage of blue native PAGE is that it does not require chromatography systems, which are expensive, but rather uses protein electrophoretic equipment that is widespread in laboratories. In terms of hands-on time, this method does not involve more work than the traditional geLC-MS/MS approach or offline chromatographic fractionation. However, as most fractionation techniques do, it has a limit to their resolution. Complexes that are very homogeneous or close in mass and shape may be beyond the resolution offered by blue native PAGE, and hence the protocol as reported here might not be universally successful in resolving distinct complexes with shared subunits. Encouragingly, we have been successful in separating two very similar tetrameric complexes sharing three subunits (M. Pardo, manuscript in preparation).
Since mass spectrometry has become increasingly sensitive, even minute amounts of non-specific interactors and contaminants can be detected in AP-MS samples. The approach presented here might aid in the discrimination of real interactors from background contamination in the affinity purification by focusing the attention on proteins with migration peaks that coincide with bait migration peaks at higher molecular weight than that of the monomeric proteins.
Several groups have used fractionation techniques followed by protein correlation profiling to delineate protein complexes at cellular scale without the need for previous isolation 10,11,12,34. However, this can result in failure to detect sub-stoichiometric interactions. The incorporation of an enrichment step through affinity purification can help to overcome this. The approach described here should be generally useful for exploring the topology of protein complexes and unraveling the multiple complexes a given protein takes part in within the same cellular context. The strategy is simple and amenable to laboratories that may not have expensive chromatographic fractionation equipment to resolve protein complexes.

In cells, most proteins perform their functions through transitory protein-protein interactions or by forming stable protein assemblies. Characterizing protein interactions is crucial for fully understanding cellular processes. Affinity purification in combination with mass spectrometry (AP-MS) is one of the most commonly applied strategies to identify native protein interactions. Significant improvements in instrument capabilities achieved in the last decade have made this approach extremely powerful. It is important to note that the interactions identified by AP-MS experiments include a mixture of direct and indirect associations between bait and preys. In addition, often proteins take part in several different complexes within the same cellular context, which might have different biological roles, and therefore the interactors that are identified by AP-MS might represent a mix of distinct protein assemblies or functional entities. It is not possible to derive such topological information a priori from the mono-dimensional lists of proteins generated by simple AP-MS experiments. However, the technique can be exploited further to define the architecture of protein complexes by combining it with one or more methods to resolve these assemblies.
In order to resolve the topology of protein interactions identified through AP-MS, several strategies have been applied. One approach is to perform iterative AP-MS experiments using the preys identified in a previous round of experiments as baits 1. Although very informative, this is quite a labor intensive task both experimentally and analytically. Protein cross-linking in combination with mass spectrometry is increasingly being used to derive topological information on protein complexes 2,3,4,5. However, computational analysis of cross-linked peptides still remains a challenging task and hence is the bottleneck in the workflow. The advent of MS-cleavable cross-linking reagents should facilitate mapping of the amino acid residues that are in close proximity in interacting proteins 6,7. Another alternative is to combine affinity purification with prior orthogonal separation techniques 8,9. Chromatographic fractionation by gel filtration or ion exchange, or sucrose gradient fractionation have also recently been used in combination with quantitative mass spectrometry to describe multiprotein complexes at a system-wide level, by-passing the complex isolation step 10,11,12,13. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been widely applied to investigate native protein interactions, typically those involving mitochondrial membrane protein complexes 14. This separation technique was also recently used in combination with label-free protein quantification and correlation profiling, not only on mitochondrial complexes 15,16,17, but also for unravelling other protein complexes from whole cells 18,19. We hypothesized that the combination of affinity purification with subsequent native fractionation approaches and quantitative MS should provide a useful strategy for resolving multiple protein assemblies containing a particular protein.
Here we describe a method that combines generic epitope-based affinity purification with blue native polyacrylamide gel electrophoresis of the isolated complexes, followed by quantitative mass spectrometry and protein correlation profiling, to resolve the multiple assemblies a protein might be involved in. We employ mouse embryonic stem cells where a protein of interest fused to an epitope tag is expressed from the endogenous locus to achieve close to physiological abundance and ensure efficient native complex isolation. This approach unravels the multiple interactions a protein engages in, resolving them into distinct assemblies based on their correlation profiles, whilst requiring no more work than conventional geLC-MS 20.

<table><tbody><tr><td>Protein G Dynabeads</td><td>Life Technologies</td><td>10003D</td><td></td></tr><tr><td>FLAG antibody M2</td><td>Sigma-Aldrich</td><td>F1804</td><td></td></tr><tr><td>Tween-20, protein grade, 10% solution</td><td>Millipore</td><td>655206</td><td></td></tr><tr><td>Dimethyl pimelimidate</td><td>Sigma-Aldrich</td><td>80490</td><td></td></tr><tr><td>0.2 M Triethanolamine buffer, pH 8.2</td><td>Sigma-Aldrich</td><td>T0449</td><td></td></tr><tr><td>3x FLAG peptide</td><td>Sigma-Aldrich</td><td>F4799</td><td></td></tr><tr><td>Vivaspin 5K NMWCO, PES</td><td>Sartorius</td><td>VS0112</td><td></td></tr><tr><td>NativePAGE 3-12% Bis-Tris gel</td><td>Life Technologies</td><td>BN1001</td><td></td></tr><tr><td>20x NativePAGE Running Buffer</td><td>Life Technologies</td><td>BN2001</td><td></td></tr><tr><td>20x NativePAGE Cathode Additive</td><td>Life Technologies</td><td>BN2002</td><td></td></tr><tr><td>4x NativePAGE sample loading buffer</td><td>Life Technologies</td><td>BN2003</td><td></td></tr><tr><td>NativeMARK</td><td>Life Technologies</td><td>LC0725</td><td></td></tr><tr><td>NativePAGE 5% G-250 sample additive&amp;nbsp;</td><td>Life Technologies</td><td>BN2004</td><td></td></tr><tr><td>Nunc conical bottom 96-well plate, polypropylene</td><td>Thermo</td><td>249944</td><td></td></tr><tr><td>Multiscreen HTS 96-well filtration system</td><td>Thermo</td><td>MSDVN6550</td><td></td></tr><tr><td>Nunc 96-well cap natural</td><td>Thermo</td><td>276002</td><td></td></tr><tr><td>TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)</td><td>Sigma-Aldrich</td><td>646547</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I1149</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher Scientific</td><td>A/0627/17</td><td></td></tr><tr><td>Trypsin, sequencing grade</td><td>Roche</td><td>11418475001</td><td></td></tr><tr><td>MaxQuant (software)</td><td>N.A.</td><td>N.A.</td><td>http://www.biochem.mpg.de/5111795/maxquant</td></tr><tr><td>Perseus (software)</td><td>N.A.</td><td>N.A.</td><td>http://www.biochem.mpg.de/5111810/perseus</td></tr></tbody></table>

resolving affinity purified protein complexes by blue native page and protein correlation profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

The workflow of the Affinity purification Blue native protein Correlation profiling by Mass Spectrometry (ABC-MS) strategy is depicted in Figure 7. Native protein complexes around a protein of interest are isolated by affinity purification using antibodies against an epitope tag (in this case FLAG) and competitive elution. The complexes are resolved by BN-PAGE, and the whole gel lane is excised into 48 sections, and prepared for shotgun LC-MS/MS. Quantitative MS information is used to generate a migration profile for each identified protein across the blue native separation. Proteins that interact to form a distinct complex display similar migration profiles with superimposable peaks. When a protein of interest takes part in more than one assembly, multiple peaks are observed in its migration profile, given the sub-complexes are within the resolving power of the blue native gel. A systematic comparison of the migration profiles can be achieved by protein correlation profiling using hierarchical clustering. The dendrogram and the peak intensities for all fractions are visualized in a heat-map, facilitating the identification of interacting proteins that belong to distinct protein complexes (Figure 8).
We used this strategy to analyze the interacting partners of Mta2, a core subunit of the NuRD chromatin remodeling complex 20. As shown in Figure 9, the migration profile of Mta2 displayed two peaks of distinct intensities between 700 kDa and 1.2 MDa, with the lower mass peak displaying higher abundance. Other NuRD core subunits, including Mta1/3, Hdac1/2 and Mbd3, showed identical separation pattern, albeit the peaks for some of the subunits, namely Chd4, Gatad2a/b and Rbbp4/7, had the inverse abundance distribution (Figure 9A, B). In contrast, the profile of Cdk2ap1, a regulatory factor that recruits the NuRD complex to Wnt gene promoters 23, only displayed the higher mass peak (Figure 9C), and so did Sall4, a transcriptional repressor that has also been shown to bind NuRD 20,24,25. Thus, fractionation of affinity purified Mta2-associated proteins by blue native PAGE was able to resolve two different forms of the NuRD complex.
ABC-MS also allows the identification of novel interactors whilst assigning them to particular protein entities. Through examination of the proteins clusters and fraction intensities represented in a heat-map we detected a strong correlation between NuRD subunits and Wdr5, a regulatory subunit of the MLL methyltransferase complex 26, with Wdr5 displaying two migration peaks coincident with the two NuRD peaks (Figure 8), suggesting a novel interaction between Wdr5 and NuRD. We confirmed this interaction by co-immunoprecipitation and co-migration in size exclusion chromatography 20.
The choice of distance metric calculation in the hierarchical clustering will influence the shape of the clusters and hence the correlations. We recommend experimenting with the different metrics to achieve the best fit with existing interaction knowledge of the protein or complex of interest. Generally we achieved best results with the Manhattan (L1) distance metric 20. Pearson correlation or Euclidean distance metrics have also been reported for identifying complexes based on alternative fractionation techniques 10,11,12.
The quantitative MS data obtained from the blue native fractions can also be used to determine the stoichiometry of protein complexes. The iBAQ (intensity based absolute quantification) value provides a measure of relative abundance of the identified proteins 27,28. The iBAQ values for the protein complex members in all the fractions within a migration profile peak are normalized to that of the bait protein to obtain relative amounts. The normalized iBAQs across a profile peak for a given protein complex member should follow a horizontal trend, and the trend values reflect the stoichiometry of the interacting proteins relative to the bait protein. For a more detailed representation of stoichiometry calculations, see 20.
<img alt="Figure 7" src="/files/ftp_upload/55498/55498fig7.jpg" /> 
Figure 7: Schematic workflow summarizing the ABC-MS strategy. This figure is modified from 20. <a href="http://cloudflare.jove.com/files/ftp_upload/55498/55498fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55498/55498fig8.jpg" /> 
Figure 8. Hierarchical clustering of BN-PAGE migration profiles of Mta2 interactors. Mta2-associated proteins were clustered based on the similarity of their migration profiles. Only a subset of the heat-map containing the NuRD complex is shown (enclosed in the blue box). The yellow box highlights the strong correlation of Wdr5 with the NuRD complex. The annotated molecular weights were estimated from the migration distances of protein standards run in the same gel. This research was originally published in Molecular and Cellular Proteomics 20 the American Society for Biochemistry and Molecular Biology. <a href="http://cloudflare.jove.com/files/ftp_upload/55498/55498fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/55498/55498fig9.jpg" /> 
Figure 9. BN-PAGE migration profile of NuRD subunits and associated proteins. A) Mta2 and NuRD subunits present at a similar intensity pattern. B) Mta2 and NuRD subunits with the inverse intensity pattern. C) Mta2 and Cdk2ap1, which is present only in the higher molecular weight NuRD entity. The annotated molecular weights were estimated from the migration profile of the protein standards. This research was originally published in Molecular and Cellular Proteomics 20 the American Society for Biochemistry and Molecular Biology. <a href="http://cloudflare.jove.com/files/ftp_upload/55498/55498fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental File" src="/files/ftp_upload/55498/proteinGroupsDB04.jpg" /> 
Supplemental data: Sample dataset. MaxQuant-derived proteingroups.txt file containing protein identifications and quantification values from an ABC-MS experiment. This research was originally published in Molecular and Cellular Proteomics 20 the American Society for Biochemistry and Molecular Biology. <a href="https://www.jove.com/files/ftp_upload/55498/proteinGroupsDB04.txt" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling at JoVE.com

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

resolving-affinity-purified-protein-complexes-blue-native-page

Research

JoVE Journal

Biochemistry

13.8K Views.  Wellcome Trust Sanger Institute.  Hier presenteren we protocollen voor affiniteitszuivering van eiwitcomplexen en hun scheiding door blauwe natieve PAGE, gevolgd door proteïne correlatie profilering middels label vrije kwantitatieve massaspectrometrie. Deze methode is nuttig voor interactomen in verschillende proteïnecomplexen lossen. 

Video: Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Chemistry

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Biology

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

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