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
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>
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			<td>Murashige and Skoog Basal Salts with minimal organics</td>
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			<td>M6899</td>
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			<td>Sigma-Aldrich</td>
			<td>N1641</td>
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			<td>Sigma-Aldrich</td>
			<td>K3253</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
			<td>S7653</td>
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			<td>Carl Roth</td>
			<td>2189.1</td>
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			<td>Sigma-Aldrich</td>
			<td>3609</td>
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			<td>Sigma-Aldrich</td>
			<td>S6776</td>
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			<td>D0632</td>
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			<td>Sigma-Aldrich</td>
			<td>P7626</td>
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			<td>E-64 protease inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>E3132</td>
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			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P9599</td>
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			<td>Na3VO4</td>
			<td>Sigma-Aldrich</td>
			<td>S6508</td>
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			<td>AcTEV Protease</td>
			<td>Thermo Fischer Scientific</td>
			<td>12575015</td>
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			<td>Rotiphorese&#160;Gel 30 (37,5:1)</td>
			<td>Carl Roth</td>
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			<td>TEMED</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
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			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Fischer Scientific</td>
			<td>26616</td>
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			<td>SBP Tag Antibody (SB19-C4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-101595</td>
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			<td>Goat anti-mouse IgG-HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
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			<td>Sigma-Aldrich</td>
			<td>B6916</td>
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			<td>Trypsin/Lys-C Mix, Mass Spec Grade</td>
			<td>Promega&#160;</td>
			<td>V5071</td>
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			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
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			<td>Thiourea</td>
			<td>Sigma-Aldrich</td>
			<td>T8656</td>
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			<td>Sigma-Aldrich</td>
			<td>9830</td>
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			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
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			<td>MTBE&#160;</td>
			<td>Biosolve</td>
			<td>138906</td>
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			<td>Biosolve</td>
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			<td>Biosolve</td>
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			<td>Biosolve</td>
			<td>12006</td>
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			<td>Trifluoroacetic acid&#160;</td>
			<td>Biosolve</td>
			<td>202341</td>
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			<td>Formic acid&#160;</td>
			<td>Biosolve</td>
			<td>69141</td>
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			<td>Unimax 2010 Platform Shaker</td>
			<td>Heidolph</td>
			<td>5421002000</td>
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			<td>Nylon Mesh (Wire diameter 34 &#181;M, thickness 55 &#181;M, open area 14%)</td>
			<td>Prosepa</td>
			<td>Custom order</td>
			<td></td>
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			<td>Glass Funnel, 47 mm, 300 ml</td>
			<td>Restek</td>
			<td>KT953751-0000</td>
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			<td>Filter Bottle Top 500 mL 0,2 &#181;M Pes St</td>
			<td>VWR International GmbH</td>
			<td>514-0340</td>
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			<td>Mixer Mill MM 400</td>
			<td>Retsch GmbH</td>
			<td>207450001</td>
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			<td>IgG Sepharose 6 Fast Flow</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-0969-02</td>
			<td></td>
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			<td>MoBiTec GmbH</td>
			<td>M1002</td>
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			<td>MoBiTec GmbH</td>
			<td>M513515</td>
			<td></td>
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			<td>Carl Roth</td>
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			<td>Carl Roth</td>
			<td>Y555.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Resprep 24-Port SPE Manifolds</td>
			<td>Restek&#160;</td>
			<td>26080</td>
			<td></td>
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		<tr>
			<td>Finisterre C18/17% SPE Columns 100mg / 1ml</td>
			<td>Teknokroma</td>
			<td>TR-F034000</td>
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			<td>Autosampler&#160;Vials</td>
			<td>Klaus Trott Chromatographie-Zubeh&#246;r</td>
			<td>40 11 01 740</td>
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			<td>Acclaim PepMap 100 C18 LC Column</td>
			<td>Thermo Fischer Scientific</td>
			<td>164534</td>
			<td></td>
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			<td>EASY-nLC 1000 Liquid Chromatograph</td>
			<td>Thermo Fischer Scientific</td>
			<td>LC120</td>
			<td></td>
		</tr>
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			<td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>IQLAAEGAAPFALGMBDK</td>
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			<td>Acquity UPLC system&#160;</td>
			<td>Waters&#160;</td>
			<td>Custom order</td>
			<td></td>
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		<tr>
			<td>ACQUITY UPLC HSS C18 Column, 100A, 1.8 &#181;M, 2.1 mM X 100 mM, 1/pkg</td>
			<td>Waters&#160;</td>
			<td>186003533</td>
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			<td>High-power ultrasonic cleaning baths for aqueous cleaning solutions</td>
			<td>Bandelin</td>
			<td>RK 31</td>
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			<td>Genedata Expressionist</td>
			<td>Genedata</td>
			<td>NaN</td>
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			<td>Xcalibur Software</td>
			<td>Thermo Fischer Scientific</td>
			<td>NaN</td>
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		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>NaN</td>
			<td>NaN</td>
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</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 ...

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