SW and TMV are supported by the NCRR and NHLBI at the National Institutes of Health. BSK and HS are supported by the NINDS and NIGMS of the National Institutes of Health.

EXPERIMENTAL PROCEDURES
The experimental procedure (Fig 2) consists of four parts that are described in a step-wise manner below.
Part 1: Subcloning and expression of the C-terminus of P2X2 receptors.
We have expressed the full length C-terminus of P2X2 receptors in bacteria to identify the brain proteins to which it binds.
<ol>
	<li>The C-terminus (residues 353-472) of the P2X2 receptor (Fig 1) was amplified by PCR, cloned into pGEX 4NT1 (GE Life Sciences) and verified with sequencing.</li>
	<li>The recombinant plasmid was transformed into E. Coli (BL21) for expression of recombinant proteins.</li>
	<li>Glycerol stocks of bacteria (E. coli BL21) containing the P2X2 CT-GST plasmid were scraped with a sterile pipette tip and inoculated into 5 ml of Luria-Bertani (LB) media with suitable selective marker (50 &#181;g/ml ampicillin) and incubated overnight in an orbital shaker at 37&#176;C.</li>
	<li>The cultures grown overnight were inoculated into 250 ml of LB media with a suitable antibiotic (50 &#956;g/ml ampicillin) and incubated in an orbital shaker at 250 rpm for 2-3 hrs at 37&#176;C until the optical density at 600 nm reached ~ 0.6-0.7.</li>
	<li>The culture was induced with 1 mM of IPTG and further incubated at 37&#176;C for 3 hrs for protein expression.</li>
	<li>The culture was then spun at 5000 g for 15 min at 4&#176;C (1ml of culture was saved for analysing the expression level and labelled as &#8216;induced&#8217;). Supernatant was discarded and the pellet was resuspended in 20 ml of Saline Tris-EDTA (STE) buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA pH 8.0).</li>
	<li>The suspension was spun at 5000 g for 15 min in a 50 ml falcon tube. The supernatant was discarded and the semi-dry pellet was frozen at -70&#176;C overnight.</li>
	<li>The -70&#176;C pellet was resuspended in 40 ml of ice cold lysis buffer (2% (w/v) sarkosyl, 15 mg lysozyme, 150 mM NaCl, 50 mM Tris pH7.5, 5 mM DTT and complete protease inhibitor tablet), and the suspension was incubated on ice for 30 min. During this incubation, 3 ml Glutathione&#8211;Sepharose 4B beads were washed with 20 ml of Phosphate Buffer Saline (PBS) and 20 ml T-PBS (0.1% (v/v) tritonX-100 in PBS) followed by PBS. The beads were resuspended in 1 ml PBS.</li>
	<li>The bacterial suspension was freeze-thawed 3 times in liquid nitrogen and sonicated (1s on/off, 30 microns for 3 min on ice) and further incubated for 30 mins with 4% (v/v) triton X-100, 10 mM MgSO4 and 2 mM ATP on ice.</li>
	<li>The lysate was spun at 20000 g for 15 min at 4&#176;C and pellet was discarded (a sample from pellet and 1 ml of supernatant was saved for SDS page to test the amounts of proteins in cytosol and membranes). The supernatant was incubated with the beads for 1 hr (or overnight) at 4&#176;C.</li>
	<li>The beads were spun at 500 g for 5 min and the supernatant was discarded (sample was saved for unbound fraction). The beads were washed with T-PBS five times (washes were saved for wash controls).</li>
	<li>The beads were then resuspended in PBS to give 50% (w/v) slurry and stored in fridge at 4&#176;C until use. The protein concentration was estimated by Bradford assay (per manufacturer&#8217;s instructions).</li>
</ol>
Part 2: Preparation of the whole brain lysates
P2X2 receptors are known to be abundantly expressed in the brain8. In the present analyses, we sought to identify the proteins interacting with P2X2 receptors from rat whole brain lysates (Fig 3).
<ol>
	<li>Adult rat brain was harvested (animals were sacrificed according to a UCLA IAACUC-approved protocol and according to the NIH Guide for the Care and Use of Laboratory Animals) and rinsed immediately ice cold PBS (3X).</li>
	<li>To lyse the cells 10 ml (~5 times the wet weight of harvested brain) of ice cold lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 5 &#956;g/ml of leupeptin, 1% (v/v) NP-40, and a protease inhibitor cocktail tablet was added to the harvested brain.</li>
	<li>Brain tissue was homogenised on ice with a glass Dounce homogenizer until a mixture of homogeneous consistency was achieved.</li>
	<li>Cell lysate was spun at 2000 g for 5 minutes to remove the unbroken cells. The supernatant was collected and spun again at 29000 g for 60 minutes to remove intact organelles and membrane aggregates.</li>
	<li>Immediately after centrifugation the supernatant was transferred to a clean tube. The protein concentration of the whole brain lysate was measured by Bradford assay (usually ~15 mg/ml).</li>
	<li>The lysate was aliquoted and stored at -70&#176;C.</li>
</ol>
Part 3: Isolation of P2X2 C-tail associated proteins
To identify the binding partners of P2X2 CT-GST from whole brain lysates, a pull down assay was performed by using CT-GST immobilised on glutathione sepharose 4B beads as bait. For controls, GST alone bound to beads was used.
<ol>
	<li>Brain lysate was precleared with GST beads for 1 hr at 4&#176;C. The pellet was discarded and the supernatant was used for further analyses.</li>
	<li>Equal amounts of GST and P2X2 CT-GST (10 &#956;g) bound to glutathione sepharose 4 B beads were incubated overnight with 100 &#181;g of precleared solubilised proteins from rat whole brain lysate, with rotation at 4&#176;C in the lysis buffer.</li>
	<li>Beads were spun at 1000 g for 5 min and the supernatant was discarded.</li>
	<li>The beads were washed once with the lysis buffer and boiled in modified Laemli buffer [4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 2% (v/v) glycerol, 0.004% (w/v) bromophenol blue and 0.125 M TrisCl pH 6.8] for 5 min.</li>
	<li>The samples were spun at 5000 g for 5 min and the supernatant separated by 10% SDS-PAGE (Fig 4). Gels were stained with SYPRO&#174; Ruby protein gel stain (according to the manufacturer&#8217;s instructions) and visualized under ultraviolet light. P2X2 CT-associated proteins were compared with those recovered using GST-null as bait. Four biological replicates were performed (i.e. four brains and four independent pull downs).</li>
</ol>
Part 4: Identification of proteins.
Proteins separated by gel electrophoresis were excised from the gel and identified by mass spectrometry12. Note: absence of dust throughout the process is critical to reduce keratin contamination. The experimenter should wear a face mask, hair net and gloves at all times and never touch the gel region of interest without the use of gloves.
<ol>
	<li>All visible protein bands recovered from the P2X2 pull down were excised with a clean razor blade and diced (Fig 4). Corresponding regions of the gel in the GST-null pull down lane were also excised in the same manner without regard to band staining (bands specific for the GST-null bait were also excised and identified) to insure that presence of proteins below the level of staining sensitivity was not missed.</li>
	<li>The gel pieces were incubated with 200 &#956;l of 50 mM ammonium bicarbonate in 50% (v/v) acetonitrile (ACN) and the tubes vortexed for 15 min at RT. Repeat this wash step.</li>
	<li>The gel pieces were dehydrated by adding 200 &#956;l acetonitrile (gel pieces should shrink and become opaque in colour).</li>
	<li>Acetontrile was removed and the gel pieces dried in a speedvac for ~10 min.</li>
	<li>The gel pieces were then incubated with 30 &#956;l of the freshly prepared 10 mM DTT/10 mM Tris (2-carboxy-ethyl) phosphine hydrochloride (TCEP) of sufficient volume to immerse the pieces. This was left for 30 min at 56&#176;C water bath.</li>
	<li>Next the DTT solution was replaced with 100 &#956;l 500 mM ammonium bicarbonate in 50% acetonitrile solution and the gel slices washed with ammounium bicarbonate.</li>
	<li>The washing solution was aspirated and 200 &#956;l of acetonitrile added to dehydrate the gels.</li>
	<li>Acetonitrile was then removed and 100 &#956;l freshly prepared 100 mM iodoacetamide solution was added to alkylate free sulfhydryls. This step proceeded for 25 min at room temperature in the dark.</li>
	<li>Iodoacetamide solution was removed and the gel pieces were washed with 200 &#956;l of 50 mM ammonium bicarbonate in 50% acetontrile (pH 8.0) for 2 min while vortexing. This wash step was repeated.</li>
	<li>After removing the wash solution, the gel pieces were incubated with 200 &#956;l acetonitrile which was then removed by speedvac for ~10min.</li>
	<li>At this step the gel pieces are ready for trypsin digestion. Gel pieces were incubated in 30 &#956;l trypsin solution (20 ng/&#956;L working concentration) and placed on ice for 10 min for the gels to be well swollen. Excess trypsin was removed and rehydrated gel particles overlaid with 30 &#956;l with 50 mM ammonium bicarbonate to keep them immersed throughout digestion which was allowed to proceed for 12 to 16 hours at 37&#176;C.</li>
	<li>Five &#956;l of 5% (v/v) Formic acid is added to deactivate the trypsin and arrest digestion.</li>
	<li>Tubes were vortexed for ~15 min and centrifuged briefly to bring the liquid to the bottom of the tube which was then transferred to a clean and labeled 0.5 ml vial.</li>
	<li>Next 30 &#956;l of 0.1% (v/v) Formic acid in 50% acetonitrile was added to gel pieces so they were just covered followed by vortexing twice for 10-15 min, separated by brief centrifugation. This step was repeated once, replacing the formic acid-acetonitrile between steps.</li>
	<li>The final liquid was removed and all extraction solution combined in a single vial. This volume was reduced to ~30 uL in the speedvac. The sample was now ready for reverse phase nano-liquid chromatography and protein identification by mass spectrometry. In this specific example, this step is carried out on an Orbi-trap tandem mass spectrometer from ThermoFisher (chosen for its high mass accuracy, fast duty cycle for protein detection and generally robust operating features), although other models and manufacturers&#8217; instruments could easily be coupled with the approach described to this point.</li>
	<li>The details and criteria for mass spectrometric analyses are now briefly presented. All peptides were separated by reverse phase nano-LC (Eksigent) and peptides were loaded onto a C18 reverse-phase column at a flow rate of 3 &#956;l/min. Mobile phase A was 0.1% formic acid and 2% ACN in water; mobile phase B was 0.1% formic acid and 20% water in ACN. Peptides were eluted from the column at a flow rate of 220 nl/min using a linear gradient from 5% B to 50% B over 90 min, then to 95% B over 5 min, and finally keeping constant 95% B for 5 min. Spectra were acquired in data-dependent mode with the Orbi-trap used for MS scans and LTQ for MS/MS scans. Peptides were identified by searching the spectra against the rat IPI database v.3.53 using the SEQUEST algorithm integrated into the Bioworks software package. Each peptide met the following criteria: XCorr &#8805;2 (+1), &#8805;3 (+2), &#8805;4 (+3), and DeltaCN&#62;0.1. All spectra used for protein identification were manually inspected and no proteins accepted when less than two peptides were identified. Only proteins present following each of the four P2X2 pull downs and absent during GST-null pull downs were considered for further analyses.</li>
</ol>
<img alt="figure 1" src="/files/ftp_upload/1178/1178fig1.jpg" />
Figure 1. Schematic representation of a P2X2 receptor subunit. A. The cartoon illustrates the topology of a P2X2 receptor subunit. The cytosolic domain consists of N and C termini. The C-terminus of P2X2 receptor (red) was used as bait for pull down assay. B. Amino acid sequence of the P2X2 receptor C-terminus used in this study.
<img alt="figure 2" src="/files/ftp_upload/1178/1178fig2.jpg" />
Figure 2. Flow chart and time line of the protocol used for expression, purification, pull down and identification of proteins. We show the outline of the protocol with the time line. Adult rat brain lysate was prepared fresh immediately before use for the pull down assays.
<img alt="figure 3" src="/files/ftp_upload/1178/1178fig3.jpg" />
Figure 3. Schematic representation of the binary proteomic analysis of P2X2 C-terminus network in the rat brain. The C-terminus of P2X2 receptors fused with GST protein bound to GST beads was used for pull down assays. Brain lysate was prepared and proteins were incubated with the immobilized recombinant proteins. Unbound fraction was washed with the lysis buffer. Proteins were identified by mass spectrometry.
<img alt="figure 4" src="/files/ftp_upload/1178/1178fig4.jpg" />
Figure 4. Identification of binding partners of the C-terminus of P2X2 receptors. Sypro stained gel showing a spectrum of putative proteins that interact with the C-terminus of receptors fused with GST (blue box). Controls lanes for GST alone (yellow box) and glutathione sepharose beads alone are also shown. The arrows indicate examples of unique bands that were excised for further analysis by mass spectrometry.

<ol>
	<li>Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion channels of excitable membranes. , (2001).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors+and+ATP+signalling+at+synapses.">Molecular physiology of P2X receptors and ATP signalling at synapses.</a> Nat Rev Neurosci. 2, 165-174 (2001).</li><li>Egan, T. M., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+calcium+ions+to+P2X+channel+responses.">Contribution of calcium ions to P2X channel responses.</a> J Neurosci. 24, 3413-3420 (2004).</li><li>Burnashev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+permeability+of+ligand-gated+channels.">Calcium permeability of ligand-gated channels.</a> Cell Calcium. 24, 325-332 (1998).</li><li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling.">Calcium signaling.</a> Cell. 131, 1047-1058 (2007).</li><li>Levitan, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+protein+complexes+associated+with+neuronal+ion+channels.">Signaling protein complexes associated with neuronal ion channels.</a> Nat Neurosci. 9, 305-310 (2006).</li><li>Khakh, B. S., North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X+receptors+as+cell-surface+ATP+sensors+in+health+and+disease.">P2X receptors as cell-surface ATP sensors in health and disease.</a> Nature. 442, 527-532 (2006).</li><li>Roger, S., Pelegrin, P., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+P2X7+receptor+currents+and+membrane+blebbing+via+constitutive+and+dynamic+calmodulin+binding.">Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding.</a> J Neurosci. 28, 6393-6401 (2008).</li><li>North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors.">Molecular physiology of P2X receptors.</a> Physiol Rev. 82, 1013-1067 (2002).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+union+of+pharmacology.+XXIV.+Current+status+of+the+nomenclature+and+properties+of+P2X+receptors+and+their+subunits..">International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits..</a> Pharmacol Rev. 53, 107-118 (2001).</li><li>Young, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+shape,+architecture+and+size+of+P2X4+receptors+determined+using+fluorescence+resonance+energy+transfer+and+electron+microscopy.">Molecular shape, architecture and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.</a> J Biol Chem. 283, 26241-26251 (2008).</li><li>Edmondson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+epsilon+signaling+complexes+include+metabolism-+and+transcription/translation-related+proteins:+complimentary+separation+techniques+with+LC/MS/MS.">Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS.</a> Mol Cell Proteomics. 1, 421-433 (2002).</li><li>Evans, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthosteric+and+allosteric+binding+sites+of+P2X+receptors.">Orthosteric and allosteric binding sites of P2X receptors.</a> Eur Biophys J. 38, 319-327 (2009).</li></ol>

Ion channels are a major class of integral membrane proteins. They contain water filled pores that selectively permit the movement of ions down their electrochemical gradients across the plasma membrane. Ion channels gate between open and the closed states. The gating step is triggered by transmitters (e.g. ATP) in case of P2X ligand gated ion channels, or it may be regulated by interactions with other proteins. The last decade has witnessed an increase in our understanding of how P2X receptors bind ATP13, but...

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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetonitrile</td>
<td>Reagent</td>
<td>JT Baker</td>
<td>9829-02</td>
<td>&nbsp;</td>
<tr>
<td>Acrylamide</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0156</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1507-01</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine Triphosphate (ATP)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>Bradford reagent</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>B-392</td>
<td>&nbsp;</td>
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<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-24972</td>
<td>&nbsp;</td>
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<td>EMD</td>
<td>3860</td>
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<td>VWR</td>
<td>VW1474-01</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>E8145</td>
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<td>Reagent</td>
<td>EMD</td>
<td>11670-1</td>
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<td>GE Healthcare</td>
<td>17-5132-01</td>
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<td>15502</td>
<td>&nbsp;</td>
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<td>I1149</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>EMD</td>
<td>1.00547.5007</td>
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<td>Leupeptin</td>
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<td>Sigma</td>
<td>L8511</td>
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<td>62971</td>
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<td>Sigma</td>
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<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
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<td>Sodium Orthovanadate (Na3VO4)</td>
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<td>Sigma</td>
<td>S6508</td>
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<td>Nonidet P40</td>
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<td>S8820</td>
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<td>Protein standard </td>
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<td>BIO-RAD</td>
<td>161-0305</td>
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<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
<tr>
<td>Screw top vial</td>
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<td>Agilent Technologies</td>
<td>5182-0866</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
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<td>SYPRO&#174; Ruby protein gel stain</td>
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<td>BIO-RAD</td>
<td>170-3125</td>
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<td>T9284</td>
<td>&nbsp;</td>
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<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
<tr>
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<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
<tr>
<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
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<td>Glycerol</td>
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<td>EMD</td>
<td>GX0185-6</td>
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proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

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14.6K Views.  David Geffen School of Medicine, University of California, Los Angeles. We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Video: Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.