Name: 2 in 1: one-step affinity purification for the parallel analysis of protein-protein and protein-metabolite complexes
Uploaded: 2018-08-06T13:30:00.000+00:00
Duration: 8 min 23 s
Description: Watch this Scientific Journal Video about 2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes at JoVE.com

We would like to kindly acknowledge Prof. Dr. Lothar Willmitzer for his involvement in the project, productive discussions, and great supervision. We are grateful to Dr. Daniel Veyel for helping with proteomic MS measurements. We appreciate Mrs. &#196;nne Michaelis who provided us invaluable technical help with LC-MS measurements. Furthermore, we would like to thank Dr. Monika Kosmacz and Dr. Ewelina Soko&#322;owska for their help and involvement in the work on the original manuscript, and to Weronika Jasi&#324;ska for technical support.

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The authors have nothing to disclose.

The presented protocol allows parallel identification of PP and PM complexes of a target protein. From cloning to final results, the experiment can be completed in as little as 8-12 weeks. Complete AP takes about 4-6 h for a set of 12 to 24 samples, rendering our protocol suitable for mid-throughput analysis.
The protocol, despite being overall straightforward, has a number of critical steps. (i) Sufficient amount of input protein and affinity beads is crucial to reach a dynamic range of metabolite detection. Efficient cell lysis is therefore a crucial step in the procedure. Poor protein yields can be a consequence of insufficient pulverization of the material or of suboptimal lysis-buffer/material ratio. (ii) Care should be taken that used reagents are MS-friendly. Strong detergents, glycerol, or excessive amounts of salt should be avoided as they interfere with MS detection. (iii) Agarose beads should not be over-dried during washing steps, and when using a vacuum manifold it is important to apply a slow flow rate so as not to destroy the beads or affect complex stability.
There are some important possible modifications to the presented protocol: (i) We use the constitutive CaMV35S promoter to maximize the amount of bait protein. Overexpression, while very useful, can have serious effects on cell homeostasis28 and lead to the formation of physiologically irrelevant interactions. Expression of tagged proteins using native promoters and where possible in a loss-of-function background is considered superior for retrieving true biological interactors. For proteins normally not expressed in plant cell cultures, a plant background may prove necessary to identify relevant interactors. (ii) When working with membrane proteins, the lysis buffer needs to be supplemented with an MS-compatible detergent. (iii) Introduction of a second affinity-purification step could improve false-positives to true-positives ratio and eliminate the need for EV controls29. A novel tandem tag with two independent protease-cleavage sites presents an attractive alternative to the size-exclusion chromatography step added by Maeda et al. 201411, which is both laborious and time consuming.
The most serious drawback of the AP is the high rate of false positives. The reasons are numerous. Constitutive overexpression was already mentioned. Another source of physiologically irrelevant interactions, unless working with isolated organelles, is preparation of whole-cell lysates containing mixtures of proteins and metabolites from different subcellular compartments. Subcellular localization should be used to filter for true interactors. Nevertheless, the majority of false positives result from unspecific binding between proteins and agarose resins. Introduction of a second purification step, as described above, offers the best solution to the problem, however comes at the cost of time and throughput. Moreover, weaker interaction may be lost as the protocol lengthens. Another caveat of AP is that despite the comprehensive information it provides about the interactome of a target protein, differentiating between direct and indirect targets of the baited protein is impossible. Targeted bimolecular approaches are needed to confirm interactions.
AP coupled with MS-based metabolomics was used to study protein- complexes in S. cerevisiae12. This work, together with our earlier observation13 that, similarly to lipids, polar and semi-polar compounds remain bound to protein complexes isolated from cellular lysates, provided conceptual groundwork for the presented protocol. Our protocol is characterized by three unique points: (i) In contrast to the yeast work12, it demonstrates that AP is suitable for retrieving not only hydrophobic but also hydrophilic protein ligands. (ii) By introducing a three-in-one extraction protocol, a single AP can be used to study protein and metabolite interactors of the bait protein. (iii) We adapted the protocol to plant cells.
Future efforts will focus on creating a novel tandem tag with two independent protease-cleavage sites. We would also like to explore suitability of the protocol to low-abundance small molecules such as plant hormones.

The method described here aims at the identification of metabolite and protein partners of a protein of choice in near-in-vivo cellular lysate conditions. It has been speculated that many more metabolites than characterized today have an important regulatory function1. Metabolites can act as biological switches, changing the activity, functionality, and/or localization of their receptor proteins2,3,4. In the last decade several breakthrough methods, enabling identification of PMI in vivo or in near-in-vivo conditions, have been developed5. Available approaches can be separated into two groups. The first group comprises techniques that start with a known-metabolite bait in order to trap novel protein partners. Methods include affinity chromatography6, drug affinity responsive target-stability assay7, chemo-proteomics8, and thermal proteome profiling9. The second group consists of a single method that starts with a known protein in order to identify small-molecule ligands10,11.
AP coupled with MS-based lipidomics was used to analyze protein-lipid complexes in Saccharomyces cerevisiae12. As a starting point, the authors used yeast strains expressing 21 enzymes involved in ergosterol biosynthesis and 103 kinases fused to a tandem-affinity purification (TAP) tag. 70% of the enzymes and 20% of the kinases were found to bind different hydrophobic ligands, shedding light into the intricate protein-lipid interaction network.
Previously, we could demonstrate that, similarly to lipids, polar and semi-polar compounds also remain bound to protein complexes isolated from the cellular lysates13. Based on these findings, we decided to optimize the AP method published previously10,11 for plant cells and hydrophilic compounds14. For this purpose, we used TAP vectors described by Van Leene et al. 2010, successfully used in plant PPI studies15. To shorten the time required to obtain transgenic lines, we decided on Arabidopsis cell cultures. We employed a one-step methyl tert-butyl ether, (MTBE)/methanol/water extraction method, allowing the characterization of proteins (pellet), lipids (organic phase), and hydrophilic metabolites (aqueous phase)16 in a single affinity-purification experiment. EV control lines were introduced to exclude false positives, e.g. proteins binding to the tag alone. As proof of concept we tagged three (of the five) nucleoside diphosphate kinases present in the Arabidopsis genome (NDPK1-NDPK3). Among other findings, we could demonstrate that NDPK1 interacts with glutathione S-transferase and glutathione. Consequently we could prove that NDPK1 is subjected to glutathionylation14.
To sum up, the presented protocol is an important tool for characterizing protein-protein and protein-small-molecule interaction networks and constitutes a major advance over existing methods.

<table><tbody><tr><td>Murashige and Skoog Basal Salts with minimal organics</td><td>Sigma-Aldrich</td><td>M6899</td><td></td></tr><tr><td>1-Naphthylacetic acid</td><td>Sigma-Aldrich</td><td>N1641</td><td></td></tr><tr><td>Kinetin solution</td><td>Sigma-Aldrich</td><td>K3253</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>10708976001</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>MgCl2</td><td>Carl Roth</td><td>2189.1</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>3609</td><td></td></tr><tr><td>NaF</td><td>Sigma-Aldrich</td><td>S6776</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>E-64 protease inhibitor</td><td>Sigma-Aldrich</td><td>E3132</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>P9599</td><td></td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S6508</td><td></td></tr><tr><td>AcTEV Protease</td><td>Thermo Fischer Scientific</td><td>12575015</td><td></td></tr><tr><td>Rotiphorese&amp;nbsp;Gel 30 (37,5:1)</td><td>Carl Roth</td><td>3029.2</td><td></td></tr><tr><td>TEMED</td><td>Carl Roth</td><td>2367.3</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder</td><td>Thermo Fischer Scientific</td><td>26616</td><td></td></tr><tr><td>SBP Tag Antibody (SB19-C4)</td><td>Santa Cruz Biotechnology</td><td>sc-101595</td><td></td></tr><tr><td>Goat anti-mouse IgG-HRP</td><td>Santa Cruz Biotechnology</td><td>sc-2005</td><td></td></tr><tr><td>Bradford Reagent</td><td>Sigma-Aldrich</td><td>B6916</td><td></td></tr><tr><td>Trypsin/Lys-C Mix, Mass Spec Grade</td><td>Promega&amp;nbsp;</td><td>V5071</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5128</td><td></td></tr><tr><td>Thiourea</td><td>Sigma-Aldrich</td><td>T8656</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I1149</td><td></td></tr><tr><td>MTBE&amp;nbsp;</td><td>Biosolve</td><td>138906</td><td></td></tr><tr><td>Methanol&amp;nbsp;</td><td>Biosolve</td><td>136806</td><td></td></tr><tr><td>Water</td><td>Biosolve</td><td>232106</td><td></td></tr><tr><td>Acetonitrile</td><td>Biosolve</td><td>12006</td><td></td></tr><tr><td>Trifluoroacetic acid&amp;nbsp;</td><td>Biosolve</td><td>202341</td><td></td></tr><tr><td>Formic acid&amp;nbsp;</td><td>Biosolve</td><td>69141</td><td></td></tr><tr><td>Unimax 2010 Platform Shaker</td><td>Heidolph</td><td>5421002000</td><td></td></tr><tr><td>Nylon Mesh (Wire diameter 34 &amp;micro;M, thickness 55 &amp;micro;M, open area 14%)</td><td>Prosepa</td><td>Custom order</td><td></td></tr><tr><td>Glass Funnel, 47 mm, 300 ml</td><td>Restek</td><td>KT953751-0000</td><td></td></tr><tr><td>Filter Bottle Top 500 mL 0,2 &amp;micro;M Pes St</td><td>VWR International GmbH</td><td>514-0340</td><td></td></tr><tr><td>Mixer Mill MM 400</td><td>Retsch GmbH</td><td>207450001</td><td></td></tr><tr><td>IgG Sepharose 6 Fast Flow</td><td>GE Healthcare Life Sciences</td><td>17-0969-02</td><td></td></tr><tr><td>Mobicol &quot;&quot;Classic&quot;&quot; with 2 different screw caps without filters</td><td>MoBiTec GmbH</td><td>M1002</td><td></td></tr><tr><td>Filter (small) 35 &amp;micro;M pore size, for Mobicol M 1002, M1003, M1050 &amp;amp; M1053</td><td>MoBiTec GmbH</td><td>M513515</td><td></td></tr><tr><td>Variable Speed Tube Rotator SB 3</td><td>Carl Roth</td><td>Y550.1</td><td></td></tr><tr><td>Rotary dishes for rotators SB 3</td><td>Carl Roth</td><td>Y555.1</td><td></td></tr><tr><td>Resprep 24-Port SPE Manifolds</td><td>Restek&amp;nbsp;</td><td>26080</td><td></td></tr><tr><td>Finisterre C18/17% SPE Columns 100mg / 1ml</td><td>Teknokroma</td><td>TR-F034000</td><td></td></tr><tr><td>Autosampler&amp;nbsp;Vials</td><td>Klaus Trott Chromatographie-Zubeh&amp;ouml;r</td><td>40 11 01 740</td><td></td></tr><tr><td>Acclaim PepMap 100 C18 LC Column</td><td>Thermo Fischer Scientific</td><td>164534</td><td></td></tr><tr><td>EASY-nLC 1000 Liquid Chromatograph</td><td>Thermo Fischer Scientific</td><td>LC120</td><td></td></tr><tr><td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td><td>Thermo Fischer Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>Acquity UPLC system&amp;nbsp;</td><td>Waters&amp;nbsp;</td><td>Custom order</td><td></td></tr><tr><td>ACQUITY UPLC HSS C18 Column, 100A, 1.8 &amp;micro;M, 2.1 mM X 100 mM, 1/pkg</td><td>Waters&amp;nbsp;</td><td>186003533</td><td></td></tr><tr><td>High-power ultrasonic cleaning baths for aqueous cleaning solutions</td><td>Bandelin</td><td>RK 31</td><td></td></tr><tr><td>Genedata Expressionist</td><td>Genedata</td><td>NaN</td><td></td></tr><tr><td>Xcalibur Software</td><td>Thermo Fischer Scientific</td><td>NaN</td><td></td></tr><tr><td>MaxQuant</td><td>NaN</td><td>NaN</td><td></td></tr></tbody></table>

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.

In the original study, three A. thaliana NDPK genes were overexpressed in PSB-L cell suspension cultures under the control of the constitutive 35S promoter14 (Figure 1). Tandem affinity tag was fused to either carboxy- or amino-terminal end of a bait protein. The affinity-purified complexes were subjected to MTBE/methanol/water extraction16. Affinity-pulled proteins and small molecules were identified using MS (Tables S2 and S3).
To correct for false positives, blank samples were used to exclude small-molecule contaminants from the chemicals and laboratory consumables. Furthermore, metabolites and proteins that bind to either an affinity tag or resin alone were accounted for by using EV control lines.To retrieve true positives, two-tailed non-paired Student&#39;s t-test and Benjamini &#38; Hochberg false discovery rate correction was applied to identify metabolites (Table S4) and proteins (Table S5) significantly enriched in the NDPKs AP experiments (N- and C-terminally tagged NDPKs) in comparison to the EV control lines (FDR &#60; 0.1). Note that in the previous work, we used absence/presence criteria to delineate protein and small-molecule interactors.
Representative results are given for NDPK1, while metabolite data focus on dipeptides, a novel class of the small-molecule regulators studied in our group. Proteomic analysis revealed 26 putative protein partners of NDPK1. By further filtering for proteins co-localized in the same subcellular compartment as NDPK1 (cytosol), the list narrowed down to 13 putative protein interactors. Among the identified proteins were glutathione S-transferase, two elongation initiation factors, tubulin, and aconitate hydratase. Metabolomic analysis revealed four dipeptides Val-Leu, Ile-Glu, Leu-Ile, and Ile-Phe that specifically co-eluted with NDPK1 (Figure 2). Note that all four dipeptides share a hydrophobic residue in their N-terminus, suggesting shared binding specificity.
To look for known protein-protein and protein-metabolite complexes we queried 13 identified proteins and four dipeptides against the Stitch database25 (Figure 3). Several observations could be made: (i) None of the interactors was previously reported for NDPK1. (ii) APX1 ortholog was reported to interact with aldehyde dehydrogenase family member ALDH7B4, while translation initiation factor FBR12 with another translation initiation factor encoded by gene AT2G40290. (iii) The identified dipeptides have no reported protein partners. Co-eluted dipeptides were not reported earlier as associated to any retrieved plant protein. However, they play important roles in other organisms: Leu-Ile, e.g., has a neurotrophin-activating effect in a human cell line26. Note that the experiment does not allow identifying the exact topology of the system. For example, a dipeptide may interact directly with NDPK1 but may well be related to any of the co-purified proteins.
Taken together, our results show that the established procedure, employing AP together with mass spectrometry, facilitates identification of protein-protein and protein-small-molecule interactors and helps generate extensive information about the interactome of the target protein.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57720/57720fig1.jpg" /> 
Figure 1. Scheme of AP-MS workflow. (A) Preparation of a native soluble fraction from plant cell culture. (B) Next steps in the AP procedure. After loading the sample onto the column, the protein of interest (POI) fused to a TAP tag binds to the IgG antibody immobilized on the agarose beads. Washing of the column facilitates removal of unbound proteins and metabolites. After performing AcTEV cleavage, POI protein-metabolite complexes are eluted. (C) Separation of complexes into protein and metabolite fraction followed by semi-quantitative MS analysis. Part of this Figure is reproduced from Luzarowski et al. 201714. <a href="/files/ftp_upload/57720/57720fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57720/57720fig2.jpg" /> 
Figure 2. Dipeptides specifically co-eluting with NDPK1. Average intensities of four dipeptides Val-Leu (A), Ile-Glu (B), Leu-Ile (C), and Ile-Phe (D) measured in AP experiment were plotted. All four dipeptides show significant enrichment in NDPK1 samples compared to EV control (asterisks represent FDR &#60; 0.1). Error bars represent standard error for 6 measurements (3 replicates of N- and 3 of C-terminally tagged proteins). <a href="/files/ftp_upload/57720/57720fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57720/57720fig3.jpg" /> 
Figure 3. Interaction network of all molecules co-eluting with NDPK1, queried against STITCH database considering only previous experimental and database evidences (confidence &#62; 0.2). Higher confidence indicates higher chances of interaction and is calculated based on the deposited data. <a href="/files/ftp_upload/57720/57720fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table S1. MaxQuant output table &#34;parameters.txt&#34;. Table includes threshold values for identification and quantification, as well as information about the databases used. <a href="/files/ftp_upload/57720/Table_S1.xlsx">Please click here to download this file.</a>
Table S2. Information from MaxQuant output table &#34;proteinGroups.txt&#34;. Table contains a list of all identified protein groups, intensities, and additional information such as number of unique peptides and score. <a href="/files/ftp_upload/57720/Table_S2.xlsx">Please click here to download this file.</a>
Table S3. Output file containing analysis of polar metabolites. Table contains a list of all identified mass features characterized by specific m/z, RT and intensity. <a href="/files/ftp_upload/57720/Table_S3.xlsx">Please click here to download this file.</a>
Table S4. Dipeptides found in AP samples in which NDPK1, NDPK2 or NDPK3 were used as bait. Dipeptides present in blank samples were excluded from the list. Two independent lines (tagged in either N- or C-terminus) for each NDPK were run in triplicate. Student&#39;s t-test and further correction of p-value using Benjamini &#38; Hochberg method were used to determine significantly enriched interactor partners of NDPKs (FDR &#60; 0.1). Given is &#916;RT calculated in relation to the reference compounds and &#916;ppm in relation to the monoisotopic mass given in Metlin27. <a href="/files/ftp_upload/57720/Table_S4.xlsx">Please click here to download this file.</a>
Table S5. Proteins co-purified with NDPK1. Two independent lines (tagged in either N- or C-terminus) for each NDPK were run in triplicate. Student&#39;s t-test and further correction of p-value using Benjamini &#38; Hochberg method were used to determine significantly enriched interactor partners of NDPKs (FDR &#60; 0.1). <a href="/files/ftp_upload/57720/Table_S5.xlsx">Please click here to download this file.</a>

Watch this Scientific Journal Video about 2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes at JoVE.com

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

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 ...

2-1-one-step-affinity-purification-for-parallel-analysis-protein

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Biochemistry

11.3K Views.  Max Planck Institute of Molecular Plant Physiology. 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.

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

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 ...

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.

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 ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

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 ...

Biology

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 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

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

Summary

Explore More Videos

Chapters in this video

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

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

Identification of Protein Interacting Partners Using Tandem Affinity Purification

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

The MultiBac Protein Complex Production Platform at the EMBL

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Tandem Affinity Purification Assay to Study Protein-Protein Interactions