We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1. Lysate Preparation
<ol>
	<li>Grow P. aeruginosa cells in LB to the desired OD. 
	NOTE: For guidance: use &#8776; 100 ml&#160;culture/sample for stationary phase cultures and &#8776; 500 ml cultur /sample for log phase cultures (OD600nm = 0.5).</li>
	<li>Pellet by centrifugation for 20 min at 5,000 x g.</li>
	<li>Resuspend 0.5-1 g of pellet in 1 ml lysis buffer (6.7 mM MES, 6.7 mM HEPES, 200 mM NaCl, 6.7 mM KAc, DDT 1 mM, pH 7.5) and add protease inhibitor (complete mini, EDTA-free) as well as DNaseI.</li>
	<li>Lyse the cells by 3 passages through a French pressure cell, at 20,000 psi (see Materials List).</li>
	<li>Ultra-centrifuge the cell lysate at 100,000 x g for 1 hr at 4 &#176;C.</li>
	<li>Save the supernatant (go to step 2).</li>
	<li>Wash the pellet with 1 ml 1x lysis buffer by pipetting up and down.</li>
	<li>Ultracentrifuge at 100,000 x g for 1 hr at 4 &#176;C.</li>
	<li>Flash-freeze the pellet in liquid nitrogen and store at -20 &#176;C until used for the capture of membrane proteins (see step 3).</li>
</ol>
2. Removal of Free c-di-GMP and Other Nucleotides (Soluble Fraction Only)
<ol>
	<li>Wash a PD10 desalting column (see Materials List) with 10 ml of cold lysis buffer.</li>
	<li>Pour the supernatant (&#8776; 1 ml) onto the PD10 in order to remove nucleotides.</li>
	<li>Elute with 4 ml cold lysis buffer (500 &#181;l steps).</li>
	<li>Select the most concentrated fractions as determined by Bradford assay, and pool them.</li>
</ol>
3. Pellet Resuspension and Solubilization (Membrane Fraction Only)
<ol>
	<li>Resuspend the pellet in 500 to 1,000 &#181;l of 1x capture buffer (without detergent) (see Table 1). 
	NOTE: The pellet is difficult to resuspend. It is advised to first pipette up and down with a pipette to roughly resuspend the pellet, then to use a syringe 27 G to well homogenize the solution.</li>
	<li>Add 1% (w/v) n-dodecyl-&#946;-D-maltopyranoside (DDM).</li>
	<li>Incubate at 4 &#176;C for at least 2 hr (or O/N) on a rotating wheel.</li>
	<li>Ultracentrifuge at 100,000 x g for 1 hr at 4 &#176;C.</li>
	<li>Harvest the supernatant.</li>
</ol>
4. Protein Concentration Measurement
<ol>
	<li>Measure the protein concentration by Bradford assay (by BCA assay for the membrane fraction).</li>
	<li>Set the total protein concentration to 10 mg/ml.</li>
</ol>
5. Capture
<ol>
	<li>Mix 300 &#181;g of protein with 1 mM of GDP, GTP, ATP, CTP, and 20 &#181;l of 5x capture buffer (100 mM HEPES, 250 mM KAc, 50 mM MgAc, 5% glycerol, pH 7.5) (Table 1). Adjust the reaction volume to 100 &#181;l with H2O. 
	NOTE: the total volume includes the volume of the cdG-CC and c-di-GMP in case of the competition control (see step 5.3 and Table 2). 
	NOTE: all experiments were performed in 200 &#181;l 12-tube PCR strips (Thermo Scientific). 
	NOTE: for the membrane fraction, make sure to always keep the DDM concentration above the critical micelle concentration (0.01% w/v) until the protein solubilization step in 8 M Urea (Step 7.1).</li>
	<li>Incubate at 4 &#176;C for 30 min on a rotating wheel.</li>
	<li>Add 10 &#181;M cdG-CC (final concentration). 
	NOTE: include a control without cdG-CC (referred as &#8220;bead control&#8221;), and a control supplemented with 1 mM c-di-GMP (&#8220;competition control&#8221;) (see Table 2). 
	NOTE: the cdG-CC concentration can be adjusted from 1 to 10 &#181;M.</li>
	<li>Incubate at 4 &#176;C for at least 2 hr (O/N for the membrane fraction) on a rotating wheel, in the dark.</li>
	<li>After a short spin, cross-link by activation of the reactive moiety of the cdG-CC with UV light for 4 min, using a CaproBox (see Materials List, &#955; = 310 nm, Irradiance &#8805;10 mW/cm&#178;, distance from the source = 2 cm). NOTE: remove the lid of the strips prior cross-linking.</li>
	<li>Add 25 &#181;l 5x wash buffer (5 M NaCl, 250 mM Tris, pH 7.5) and 50 &#181;l of well resuspended streptavidin magnetic beads, gently homogenize.</li>
	<li>Incubate at 4 &#176;C for 1 hr on a rotating wheel.</li>
</ol>
6. Washing Steps
NOTE: (Magnet: see Materials List). Start with a capture of the magnetic beads in the PCR strip lid, with the magnet. Then replace the PCR strip by a new one containing the next washing solution. Remove the magnet and resuspend the beads, and incubate 2 min. Spin down and replace the lid by a fresh lid.
<ol>
	<li>Washing steps (soluble fraction only)
	<ol>
		<li>Wash 6 times in 200 &#181;l 1x washing buffer.</li>
		<li>Wash once in 200 &#181;l HPLC grade H2O.</li>
		<li>Wash 6 times in 200 &#181;l 80% acetonitrile.</li>
		<li>Wash 2 times in 200 &#181;l HPLC grade H2O.</li>
	</ol>
	</li>
	<li>Washing steps (membrane fraction only)
	<ol>
		<li>Wash 5 times in 200 &#181;l 1x washing buffer + 0.1% DDM.</li>
		<li>Wash 2 times in 200 &#181;l 1x washing buffer + 0.05% DDM.</li>
		<li>Wash once in 200 &#181;l 1x washing buffer + 0.025% DDM.</li>
		<li>Wash once in 200 &#181;l 1x washing buffer + 0.0125% DDM.</li>
		<li>Wash 3 times in 200 &#181;l 100 mM ABC (ammonium bicarbonate, NH4CO3) + 2 M Urea.</li>
	</ol>
	</li>
</ol>
7. MS Sample Preparation
<ol>
	<li>Resuspend the beads (directly in the lid) in 20 &#181;l 100 mM ABC (100 mM ABC + 8 M Urea for the membrane fraction) and transfer into 1.5 ml tubes.</li>
	<li>Membrane fraction only: incubate at 60 &#176;C for 5 min, shaking at 500 rpm.</li>
	<li>Add 0.5 &#181;l 200 mM TCEP (tris(2-carboxyethyl)phosphine) and incubate at 60 &#176;C for 1 hr, shaking at 500 rpm. Cool down to 25 &#176;C.</li>
	<li>Add 0.5 &#181;l of freshly prepared 400 mM iodoacetamide and incubate at 25 &#176;C for 30 min, shaking at 500 rpm and in the dark.</li>
	<li>Add 0.5 &#181;l 0.5 M N-acetyl-cysteine and incubate at 25 &#176;C for 10 min, shaking at 500 rpm.</li>
	<li>Membrane fraction only: add 1 &#181;l Lys-C and incubate at 37 &#176;C, O/N.</li>
	<li>Add 2 &#181;g trypsin and incubate O/N at 37&#176;C, shaking at 500 rpm (wrap in Parafilm to prevent drying). 
	NOTE: the samples can be stored at -20 &#176;C at this stage. 
	NOTE: membrane fraction only: add 100 mM ABC to adjust the urea concentration to &#60;2 M before addition of trypsin.</li>
	<li>Briefly spin down the tubes and collect beads with the magnet.</li>
	<li>Transfer the supernatant into a new 1.5 ml&#160;tube (repeat this step if beads are left).</li>
	<li>Add 5 &#181;l 5% TFA (trifluoroacetic acid) + 1 &#181;l 2 M HCl (15 &#181;l 5% TFA+ 5 &#181;l 2 M HCl for the membrane fraction).</li>
	<li>Condition C18 MicroSpin columns (The Nest Group, MA, USA) with 150 &#181;l acetonitrile (spin 20 sec at 2,400 rpm).</li>
	<li>Equilibrate the C18 columns 2 times with 150 &#181;l 0.1% TFA (spin 20 sec, 2,400 rpm).</li>
	<li>Load the sample and spin 2 min, 2,000 rpm.</li>
	<li>Reload the flow-through onto the column and repeat the spinning step (2 min, 2,000 rpm).</li>
	<li>Wash 3 times with 150 &#181;l 0.1% TFA, 5% acetonitrile (20 sec, 2,400 rpm).</li>
	<li>Take a new tube and elute twice with 150 &#181;l 0.1% TFA, 50% acetonitrile (2 min, 2,000 rpm).</li>
	<li>Dry the peptides in a speed-vac.</li>
	<li>Resuspend in 40 &#181;l 98% H2O, 2% acetonitrile, 0.15% formic acid.</li>
	<li>Sonicate 20 sec (pulse cycle 0.5, amplitude 100%; see Materials List) and spin down 5 sec, 12,000 rpm (benchtop centrifuge). Vortex 10 sec, and spin down 5 sec, 12,000 rpm. Transfer in a HPLC vial for LC-MS/MS analysis.</li>
	<li>Freeze at -20 &#176;C. 
	NOTE: the samples can be stored at -20 &#176;C at this stage.</li>
</ol>
8. LC-MS/MS Analysis
<ol>
	<li>Run a nano-LC (nano-LC systems) equipped with a RP-HPLC column (75 &#956;m x 37 cm) packed with C18 resin (Magic C18 AQ 3 &#956;m) using a linear gradient from 95% solvent A (0.15% formic acid, 2% acetonitrile) and 5% solvent B (98% acetonitrile, 0.15% formic acid) to 35% solvent B over 60 min at a flow rate of 0.2 &#181;l/min.</li>
	<li>Analyze the peptides using LC-MS/MS (dual pressure LTQ-Orbitrap Velos mass spectrometer, connected to an electrospray ion source). 
	NOTE: The data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 60,000 FWHM followed by MS/MS scans in the linear ion trap of the 20 most intense ions. To increase the efficiency of MS/MS attempts, the charged state screening modus was enabled to exclude unassigned and singly charges ions. Collusion induced dissociation was triggered when the precursor exceeded 100 ion counts. The dynamic exclusion duration was set to 30 sec. The ion accumulation time was set to 300 msec (MS) and 50 msec (MS/MS).</li>
</ol>
9. Database Search
<ol>
	<li>Download the P. aeruginosa NCBI-database via the NCBI homepage (<a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a>).</li>
	<li>Convert the MS raw spectra into mascot generic files (mgf) using the MassMatrix conversion tool (<a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py</a>).</li>
	<li>Search this mgf file using MASCOT version 2.3 against the P. aeruginosa NCBI-database containing forward and reverse-decoy protein entries.</li>
	<li>Perform an in silico trypsin digestion after lysine and arginine (unless followed by proline) tolerating two missed cleavages in fully tryptic peptides.</li>
	<li>Set the database search parameters to allow oxidized methionines (+15.99491 Da) as variable modifications and carboxyamidomethylation (+57.021464 Da) of cysteine residues as fixed modification. For MASCOT searches using high-resolution scans, set the precursor mass tolerance to 15 p.p.m. and set the fragment mass tolerance to 0.6 Da. Finally, set the protein FDR to 1%.</li>
	<li>Import the Mascot searches of P. aeruginosa CCMS experiments into Scaffold (Proteomesoftware, Version 3), set the parameters to obtain a protein FDR close to 1%, and extract the total spectral counts.</li>
	<li>For the representative results presented in this paper, we used a paired T test to compare the experiment with the competition control, and only considered hits with a p value below 0.1, and a spectral count ratio above 2 (experiment spectral counts/competition control spectral counts).</li>
	<li>Data can be exported in any spreadsheet software for further analysis.</li>
</ol>
10. Label-free Quantification
<ol>
	<li>Import the raw files into Progenesis LC-MS software (Nonlinear Dynamics, Version 4.0).</li>
	<li>Perform LC-MS alignment and feature detection in default settings.</li>
	<li>Export the data in mgf format from Progenesis LC-MS.</li>
	<li>Search the MS/MS spectra using the MASCOT against the NCBI P. aeruginosa database containing forward and reverse-decoy protein entries.</li>
	<li>Import the database search results into Progenesis LC-MS and map the peptides identifications to MS1 features.</li>
	<li>For data evaluation (calculation of significance levels, fold-change ratios) the ProteinSQAnalysis script of SafeQuant was used (threshold: q value below 0.1, spectral count ratio above 2).</li>
</ol>

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The authors have nothing to disclose.

Special care should be taken at several steps of the protocol. The protein concentration is a critical parameter with a concentration of 10 mg/ml being difficult to reach when cells are grown under specific growth conditions (e.g. biofilms or small colony variants). Thus, the pellet resuspension should be performed in a low volume of lysis buffer. Protein concentrations can be decreased to 8 mg/ml. Compared to the method published by Nesper et al.1, we added various nucleotides to the capture...

c-di-GMP is a key second messenger used by most bacteria to control various aspects of their growth and behavior. For instance, c-di-GMP regulates cell cycle progression, motility and the expression of exopolysaccharides and surface adhesins2-4. Through the coordination of such processes c-di-GMP promotes biofilm formation, a process which is associated with chronic infections of a range of pathogenic bacteria5. c-di-GMP is synthetized by enzymes called diguanylate cyclases (DGCs) that harbor a catalytic GGDEF domain4. Some DGCs possess an inhibitory site that down regulates the cyclase activity upon c-di-GMP binding. The degradation of c-di-GMP is catalyzed by two distinct classes of phosphodiesterases (PDEs) harboring either a catalytic EAL or HD-GYP domain6,7.
The majority of the known effector proteins that directly bind c-di-GMP belong to one of only three classes of proteins: catalytically inactive GGDEF or EAL domains and PilZ domains, small molecular switches that undergo conformational changes upon c-di-GMP binding8. DGCs, PDEs and PilZ proteins are well characterized and their domains can be predicted in silico relatively safely. A particular interest is now focused on the identification of new classes of c-di-GMP effectors. Some c-di-GMP effectors with different binding motifs were described recently such as the CRP/FNR protein family Bcam1349 in Burkholderia cenocepacia or the transcriptional regulator FleQ in P. aeruginosa9,10. In addition, c-di-GMP-specific riboswitches were recently identified and shown to control gene expression in a c-di-GMP-dependent manner11. The c-di-GMP binding motifs of different effectors are only poorly conserved making bioinformatic predictions of such proteins difficult. To address this issue, we developed a biochemical method, which is based on the use of a c-di-GMP specific Capture Compound combined with mass spectrometry 1,12,13.
We have recently engineered a novel trivalent c-di-GMP Capture Compound (cdG-CC, Figure 1)1. This chemical scaffold is composed of: 1) a c-di-GMP moiety used as bait to capture c-di-GMP binding proteins, 2) a UV-photoactivatable reactive group used to cross link the cdG-CC to the bound proteins and 3) a biotin to isolate the captured proteins using streptavidin-coated magnetic beads. The cdG-CC can be used to directly and specifically capture c-di-GMP effectors from complex mixture of macromolecules as cell lysates. Capture Compound based and chemical proteomics based approaches have previously been reported to be applicable to a wide range of organisms, e.g. Caulobacter crescentus, Salmonella enterica serovar typhimurium and P. aeruginosa1,14.
In this methodological paper, we provide an in depth description of the CCMS procedure using extracts of P. aeruginosa as an example. This study establishes CCMS as a powerful and versatile tool to biochemically identify novel components involved in small molecule signaling.

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To identify novel c-di-GMP effectors in P. aeruginosa we systematically used CCMS to analyze the soluble and membrane fractions of P. aeruginosa strain PAO1 from a log phase culture (OD600 = 0.5). Here we summarize and discuss representative results of this fishing expedition. Four independent biological replicas were used. For each experiment two different cdG-CC concentrations were used (5 &#181;M and 10 &#181;M). To probe for specificity, experiments were carried out in the presence or abs...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

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

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.

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

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Research

JoVE Journal

Biology

11.5K Views.  Biozentrum of the University of Basel. 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.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

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.

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 ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

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 ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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 ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...