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Protein small molecule interactions are at the core of cell regulation controlling metabolism and development. We reasoned that due to the lack of system wide approaches only a minority of those regulatory molecules are known. In order to see whether or not this assumption is true we developed an effective approach for the identification of small molecules having potential regulatory role that obviates the need of protein or small molecule baits. At the core of this approach is a simple biochemical co-fractionation taking advantage of size differences between proteins and small molecules. Metabolomics based analysis of small molecules co-fractionating with proteins identified a multitude of small molecules in Arabidopsis suggesting the existence of numerous, small molecules/metabolites bound to proteins representing potential regulatory molecules. The approach presented here uses Arabidopsis cell cultures, but is generic and hence applicable to all biological systems.
Recent years brought significant advances in so called omics techniques allowing for simultaneous quantification of thousands of biological molecules including transcripts, proteins, and metabolites1. These molecules present building blocks of life but it is their interactions that enable life. Large-scale analysis of molecular complexes is thus one of the next great challenges to be addressed. Among these, the analysis of protein – metabolite interactome (PMI), irrespective of its high potential importance for both basic research (i.e. identifying novel signaling molecules2) and translational research (i.e. identifying lead compounds for drugs3), received comparatively little attention. The reason for this is twofold: First, the monitoring and identification of metabolites is particularly troublesome considering the large diversity of small molecules. Second, no generally applicable approaches allowing the system wide monitoring of protein – metabolite interactions have been described. Most existing approaches can be divided into those using metabolites as baits to “fish out” interacting proteins or vice versa. Immobilizing small molecules to identify protein receptors is a straight forward approach and is being used since decades4,5. Cross linking approaches represent another way of pulling protein receptors of modified small molecules6. Conversely, several studies discovered protein binding small molecules by tagging proteins of interest2,3,7. The downside of these methods is the modification of the prey which may influence the interaction. Methods avoiding immobilization of either interaction partner exist, but are so far restricted to samples of highly reduced complexity comprising predefined proteins or small molecules8,9,10.
Here we show an alternative approach that relies on global analysis of protein bound small molecules using metabolomics technologies. The basic principle of our approach is based on the supposition that small molecules/metabolites interacting with proteins and forming stable complexes will fractionate together. Thus when applying any type of size separation (size exclusion chromatography, size filtration and the like) both should appear in the high molecular weight fraction. In contrast, non-bound metabolites will remain in the low molecular weight fraction.
To see whether or not this assumption holds true, and to obtain a first insight into the wealth of protein bound metabolites, we applied our approach to plant cell suspension culture extracts. To this end, Arabidopsis thaliana cell cultures11 were lysed by physical stress and subjected to an ultracentrifugation step (cf. Methods). The supernatant containing essentially soluble proteins and metabolites (later referred to as input) was subsequently subjected to a size filtration spin column with a 10kDa cutoff to separate proteins (high molecular weight fraction) from free metabolites (low molecular weight fraction, flow through) (Fig. 1). Subsequently the fraction retained on the filter (mostly proteins) was washed thoroughly in order to remove any non-bound metabolites (wash). To release metabolites which were non-covalently bound to proteins heat denaturation was applied (elution). All samples (input, flow through, wash and eluate) were subsequently dried and re-extracted using Methyl-tert-butyl ether (MTBE)-Methanol-Water, which separates proteins (in the pellet) from polar and lipid small molecule fractions12. For the purpose of this work, polar compounds were subsequently analyzed by LC/MS (Fig. 1). The above described filtration procedure relies on two assumptions: (1) that the protein metabolite complex is stable enough to be retained on a spin column whereas all non-bound metabolites will be in the flow-through, and (2) that upon heat denaturation metabolites not covalently bound to proteins will be released. Overall, LC/MS analysis of input, flow through, wash and eluate samples resulted in 81 metabolic features which could be annotated to a metabolite using our in-house reference compounds library (Supplementary Table S1). As expected, the flow through contained many (unbound) metabolites and the metabolite content decreased in the washing. Strikingly, after heat treatment many metabolites, while being absent in wash samples, were detectable in the eluate. This strongly suggests that this large fraction of metabolites is indeed forming stable complexes with proteins (Fig. 2). Among these were well-known ligands such as cyclic nucleotides (cGMP, cAMP, cCMP), co-factors (FAD, NAD, FMN), and dipeptides (Fig. 2, Supplementary Table S1). These results indicate that in the biological system analyzed numerous metabolites/small molecules form a non-covalent but stable complex with proteins.
To challenge our filtration procedure we addressed two major points that in our opinion could raise concern. First, heat denaturation may lead to both false positives and negatives by influencing small molecule chemical composition e.g. by promoting hydrolysis. To test this, we compared results of the elution fraction with or without heat denaturation prior to MTBE-Methanol-Water extraction. Comparison of the metabolites obtained by the two methods did not reveal major differences showing a high correlation (R=0.99) (Supplementary Fig. S1), suggesting that both approaches could be used interchangeably. Second, salt used in the lysis and washing buffers can affect interactions. Too low salt concentrations may promote unspecific binding, while too high concentrations will destroy interactions by interfering with ionic bonds and by promoting protein denaturation. To find optimal conditions we compared elution profiles obtained using three different NaCl concentrations: 0.05M, 0.5M and 1.5M. As expected high salt conditions (1.5M) were disruptive for most interactions analyzed and fewer metabolites could be found in the eluate (Supplementary Fig. S2a). Shifting from 0.05M to 0.5M NaCl had a minor effect on the number of identified metabolites, but still decreased their quantity (Supplementary Fig. S2b). Thus for consecutive experiments we used intermediate 0.15M concentration (see SEC experiments).
In a next step we decided to move to an alternative size fractionation approach. To this end we applied size exclusion chromatography (SEC) to the native protein-metabolite extract using a column which separates molecular complexes from approximately 600kDa to 10kDa13,14 (Fig. 1). The chromatogram of the absorption at 280nm indicates reproducible separation of complexes present in our input sample by SEC (Fig. 3a). As expected, the protein content in the collected fractions showed separation of protein containing molecular complexes, whereas no protein was detectable in fractions later than C12 corresponding to a molecular weight smaller than 10kDa (Fig. 3a,b, Supplementary Fig. S1). To see whether or not the protein fractions contained metabolites/small molecules which based on our first principles, could only be understood if assuming stable protein-metabolite complexes, we analyzed 57 fractions for occurrence of polar metabolites by LC/MS. According to the results obtained from size filtration we detected the majority of metabolic features as non-protein bound and therefore eluting after one total mobile phase volume of the column (Fig. 3c). In addition, and more important however we detected specific elution profiles of small molecules in fractions representing theoretical MWs between 10 to 600kDa.
Three features of small molecule elution profiles are worth commenting. Firstly, the SEC results confirmed to a large extent the results of the size filtration experiment described above. Indeed 51 of the annotated metabolites identified as binding to proteins from the size filtration experiment were also observed in the SEC data distributed along the whole separation range (Fig. 3d). Secondly, for most metabolites/small molecules we observed their occurrence in distinct fractions and not throughout all SEC fractions which is a clear indication of a specific binding to one or more proteins eluting in these fractions. Thirdly and most important for proof of concept, we observed metabolites well known to interact with proteins most notably co-factors such as FMN or NAD to display distinct peaks in the separation range indicating the presence of multiple though specific binding to protein(s) (Fig. 3e,f).
To assure that differential eluting metabolites are truly metabolite-protein complex derived we performed a control experiment with a protein-free sample. To this end, we precipitated proteins from the input sample with 80% acetone and reconstituted the small molecules in lysis buffer before applying it to the SEC column. In line with SEC separating molecular complexes based on their size we did not observe any significant differential metabolic features across the SEC fractions (Fig. 3c) but all metabolites appeared after one total mobile phase volume as expected for free small molecules.
Thus the data presented up to here show elution of a small proportion of metabolites present in a cell free extract in the high-molecular weight fraction which is unexpected whereas the by far largest part of metabolites elute in the low-molecular weight area which is expected. Furthermore the elution of metabolites in the high-molecular weight fraction is dependent on the presence of proteins. These observations together with the filtration experiments described above suggest that the metabolites appear in the high-molecular weight fraction because they are bound to proteins.
If this is true and if this approach is going to be useful to monitor system-wide protein metabolite complexes one would request that known metabolite-protein interactions should at least partially be covered. To this end we performed two types of experiments: In a first experiment we tested all protein fractions via an antibody for the presence of a given protein. Glutamine Synthetase (GLN) is well-known to bind glutamate. To see if GLN is migrating together with one of the glutamate peaks, we probed the fractions of SEC with an antibody against GLN in a Western blot experiment. As evident from Supplementary Figure S4, the major peak of GLN coelutes with one of the peaks of glutamate. This major GLN peak appeared from the SEC column at approximately 390kDa and hence likely represents the homodecamer of GLN15. Several other proteins which in addition were recognized by the antibody may represent another GLN isoform or cross-reacting proteins (Supplementary Figure S4). Pyridoxal 5′-phosphate and Flavin mononucleotide (FMN) represent two well-known co-factors. Their elution profiles in the SEC experiment display the highest intensity in the high molecular weight area in fractions B05 and C1 respectively (Fig. 3e,f). In a second experiment we thus performed a proteomics analysis of these two fractions to see whether or not we can detect proteins which are known to bind either of the two co-factors (Supplementary Table S5). Within the proteins detected in fraction B05 (a total of 203 proteins) 8 proteins were detected which are described to interact with Pyridoxal 5′-phosphate (Fig. 4 for the network of these proteins as based on the STITCH16 database) whereas in fraction C1 (where we detected a total of 133 proteins) we did not detect any protein interacting with Pyridoxal 5-phosphate but did detect one protein known to interact with FMN (Fig. 4) again based on the STITCH database. Thus taken together these experiments demonstrate that metabolites respectively co-factors coelute with proteins known to interact with these proteins thus strongly lending further support to the validity of our approach.
In summary, we provide evidence that biological systems (here: Arabidopsis thaliana suspension cultures) contain a multitude and up to now unexplored number of small molecules/metabolites which form stable complexes with proteins and thus represent candidates of small molecules displaying a signaling/regulatory function. The approach described here will largely improve the identification of novel metabolite-protein interactions by its (1) dispensability of protein and metabolite baits (2) proteome/metabolome wide scale (3) relative simplicity and (4) generic nature making it suitable across biological systems. Nevertheless, our method is not free of limitations. As with other techniques starting with a native lysate there is always a chance of false positives related to mixing proteins and metabolites present in separate organelles and/or used buffering conditions. Obtained binding may be therefore specific in our experiment but not necessarily biologically relevant. Moreover, co-elution data obtained from SEC provide indication of binding event but require independent validation.
Arabidopsis cells cultures11 were grown in MSMO medium (Sigma Aldrich) supplemented with 3% sucrose, 0.05mg/L kinetin and 0.5mg/L 1-naphthaleneacetic acid on orbital shaker at 130rpm in the light. Cells were passaged weekly to fresh medium and harvested during logarithmic growth using rapid filtration and liquid nitrogen snap freezing.
Frozen cells were grinded with mortar and pestle or a Retsch mill (Retsch GmbH, Haan, Germany) for 4 times 1min at 30rps. 1.5mL (for size filtration) or 0.7mL (for size exclusion chromatography) of lysis buffer (50mM Tris-HCl pH 7.5, 500mM NaCl, 1.5mM MgCl2, 5mM DTT, 1mM PMSF, 1x Protease Inhibitor Cocktail (Sigma-Aldrich), 0.1mM Na3VO4 and 1mM NaF) were added per 1g of cells. In SEC experiments, 50mM ammonium bicarbonate-HCl pH7.5 was used instead of Tris-HCl as buffering agent and the NaCl content was lowered to 150mM. After thawing on ice the extract was filtered through miracloth and subsequently centrifuged 10min at 3452g, 4°C. Ultracentrifugation 45min at 35000rpm (max 165052g, avg 125812g), 4°C was used to prepare the soluble fraction.
2.5–3mL of soluble fraction (see above) were filtered using Amicon 10kDa Ultra centrifugal filter units (Millipore). At this stage 400μL aliquots of input and flow through were kept for metabolic analysis. Two washing steps, first using 5mL and second 1.5mL of wash buffer (50mM TrisHCl pH 7.5, 500mM NaCl, 1 196.5mM MgCl2) were applied to get rid of the remaining free metabolites. Approximately 1.5mL of wash buffer was added to the column to cover the filter and a 10min, 100°C treatment was used to denature proteins and to dissociate protein-metabolite complexes. 1mL–1.2mL aliquots from second wash step and eluate were dried and kept for metabolic analysis. Centrifugation steps were performed at 3452g for 15–30minutes.
2.5mL of soluble fraction corresponding to 50mg of protein were used for the separations. SEC was performed with a HiLoad 16/600 Superdex 200 prep grade column (GE Healthcare Life Science, Little Chalfont, UK) connected to an ÄKTA explorer 10 (GE Healthcare Life Science, Little Chalfont, UK) operating at 4°C. The flow rate was set to 0.8mL/min. 57 fractions of 1.5mL were collected from 40 to 125.5mL elution volume of which 1mL was dried in a speed-vac overnight and stored at −80°C for metabolomic analysis. For the protein free control experiment, 50mg of protein of the soluble fraction was precipitated with 80% acetone at −20°C for 5h. After pelleting denatured proteins by centrifugation at 3452g for 20min at 4°C, the supernatant was dried overnight in a speed-vac. Small molecules were resuspended the next day in the original volume of lysis buffer and used for SEC.
Samples were extracted as described by12. This method uses a Methyl tert-butyl ether (MTBE)/Methanol/Water solvent system to separate proteins, lipids, and polar compounds into pellet, organic, and aqueous phase, respectively. After extraction, the aqueous phase was dried in a speed-vac and stored at −80°C until L C/MS analysis. Samples were measured using ultra-performance liquid chromatography coupled to an Exactive mass spectrometer (Thermo-Fisher; http://www.thermofisher.com) in positive and negative ionization mode as described in ref. 12. Processing of chromatograms, peak detection, and integration were performed using REFINER MS 9.0.4 (GeneData; http://www.genedata.com). Processing of mass spectrometry data included retention time alignment, peak detection, removal of the isotopic peaks, as well as chemical noise. Obtained metabolic features (m/z at a given retention time) were queried against an in-house reference compound database allowing 0.15min retention time and 3ppm m/z deviation. For metabolites that showed a higher ppm error in standard compound measurements we allowed a corresponding deviation of up to 10ppm (as indicated in the Supplemental Tables).
LC/MS peak lists obtained as Refiner MS output were subsequently analyzed using EXCEL (Microsoft Corp.) or R (http://www.r-project.org/). Heatmaps were produced using the heatmap package in R with default settings on appropriately transformed data. All other plots were done with the basic plotting function in R. Figures were post processed with Adobe Illustrator (Adobe Systems) when needed.
Pellets recovered after metabolite extractions were used for mass spectrometric protein analysis. Proteins were dissolved in 100μL of 6M urea/2M thiourea in 40mM ammonium bicarbonate buffer. An equivalent to 100μg protein diluted to 46μL was treated with 5mM dithiothreitol for 30min at RT, followed by cysteine alkylation with 15mM iodacetamide for 20min at RT in the dark. Proteins were digested using a LysC/Trypsin Mix (#V5072, Promega) according to the manufacturer instructions. After digest the samples were finally acidified to pH<2 by adding trifluoroacetic acid. Desalting was done on C18 cartridges (SPE Columns C18/17%; #TR-F034000, Finisterre) according the manual instructions and concentrated to dryness in a speed vac. Peptides were analyzed on an EASY-nLC1000 system (Thermo Fischer Scientific) connected to a LTQ Orbitrap XL mass spectrometer (Thermo Fischer Scientific) as detailed below. Buffer A consisted of 3% acetonitrile, 0.1% formic acid and Buffer B of 63% acetonitrile, 0.1% formic acid. Dried peptide samples were resuspended in 50μL Buffer A and 2μL were separated in a 90min gradient using an Acclaim® PepMap RSLC analytical column (C18, 2μm, 100Å, 75μm i.d.×150mm, #164534, Thermo Fischer Scientific). The gradient started from 3% to 15% acetonitrile in 30min, to 30% acetonitrile over 60min followed by a 10min washout with 60% acetonitrile. The LTQ Orbitrap operated with a data dependent top 6 method as follows: MS full scans were performed in FTMS with resolution set to 60000, from 300.0 to 1600.0m/z, a maximum fill time of 250msec and an AGC target value of 1E6 ions. A maximum of 6 data dependent MS2 scans were performed in the ion trap set to an AGC target of 1E4 ions with a maximal injection time of 100msec. Precursor ion fragmentation was achieved by collision induced fragmentation with a minimal signal of 250, isolation width of 2m/z, normalized collision energy of 35, activation Q 0.25 and activation time 30. Charge states of 1 were rejected. Raw data were analyzed with MaxQuant version 1.5.6.017 and its build-in search engine Andromeda18 using the default settings. The Arabidopsis protein database was downloaded from from Uniprot (http://www.uniprot.266org/proteomes/UP000006548) containing 31423 proteins, last modified May 14, 2016. The MaxQuant output is shown in Supplemental Table 5. To identify fraction specific proteins we required proteins to be present in the two replicates of one fraction, but to be absent in the respective other fraction.
Proteins in fractions of one of the SEC experiments were analyzed by SDS-PAGE (10% Acrylamide). For this samples were loaded based on equal volume (15μL+3μL 5x loading buffer). Proteins were blotted on a membrane and incubated with the primary antibody against Glutamine Synthetase (Anti-GLN1 GLN2 antibody Antisera (AS08295), 1:1000) in 5% Milk-TBST for 1h at room temperature. The secondary antibody (goat anti-rabbit IgG –HPR, Antisera (AS09602), 1:20 000) was incubated for another hour at room temperature in 5% Milk-TBST. HRP signal was detected using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher) as describe in the manual and exposed for 5min.
How to cite this article: Veyel, D. et al. System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants. Sci. Rep. 7, 42387; doi: 10.1038/srep42387 (2017).
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We thank the group of Dr. Mark Aurel Schöttler providing the Glutamine Synthetase antibody.
The authors declare no competing financial interests.
Author Contributions S.K. was instrumental in devising experimental strategy, D.V., A.S., M.K., executed all the experiments and analyzed data, E.S. performed the Western blots, A.M., M.L., J.S. provided technical support, A.S. and L.W. coordinated the project.