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 ...

capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Research

JoVE Journal

Biology

11.6K 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 ...

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 ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...