This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2019-04658); NSERC Discovery Launch Supplement for Early Career Researchers (DGECR-2019-00081) and by McGill University. &#160;

1. Analysis of IP- or PI-binding proteins by affinity chromatography and Western blotting
<ol>
	<li>Cell growth, lysis and affinity chromatography
	<ol>
		<li>Grow T. brucei cells to mid-log phase and monitor cell viability and density. A total of 5.0 x 107 cells is sufficient for one binding assay.
		<ol>
			<li>For bloodstream forms, grow cells in HMI-9 media supplemented with 10% fetal bovine serum (FBS) at 37 &#176;C and 5% CO2. Keep cell density between 8.0 x 10...

The identification of proteins that bind to IPs or PIs is critical to understand the cellular function of these metabolites. Affinity chromatography coupled to Western blot or mass spectrometry offers an opportunity to identify IP or PI interacting proteins and hence gain insights on their regulatory function. IPs or PIs chemically tagged [e.g., Ins(1,4,5)P3 chemically linked to biotin] and crosslinked to agarose beads via streptavidin or captured by streptavidin magnetic beads allows the isolation of interacting protein...

Inositol phosphates (IPs) and phosphoinositides (PIs) play a central role in eukaryote biology through the regulation of cellular processes such as the control of gene expression1,2,3, vesicle trafficking4, signal transduction5,6, metabolism7,8,9, and development8,10. The regulatory function of these metabolites results from their ability to interact with proteins and thus regulate protein function. Upon binding by proteins, IPs and PIs may alter protein conformation11, catalytic activity12, or interactions13 and hence affect cellular function. IPs and PIs are distributed in multiple subcellular compartments, such as nucleus2,3,14,15, endoplasmic reticulum16,17, plasma membrane1 and cytosol18, either associated with proteins3,19 or with RNAs20.
The cleavage of the membrane-associated PI(4,5)P2 by phospholipase C results in the release of Ins(1,4,5)P3, which can be phosphorylated or dephosphorylated by IP kinases and phosphatases, respectively. IPs are soluble molecules that can bind to proteins and exert regulatory functions. For example, Ins(1,4,5)P3 in metazoan can act as a second messenger by binding to IP3 receptors, which induces receptor conformational changes and thus release of Ca2+ from intracellular stores11. Ins(1,3,4,5)P4 binds to the histone deacetylase complex and regulates protein complex assembly and activity13. Other examples of IPs regulatory function include the control of chromatin organization21, RNA transport22,23, RNA editing24, and transcription1,2,3. In contrast, PIs are often associated with the recruitment of proteins to the plasma membrane or organelle membranes25. However, an emerging property of PIs is the ability to associate with proteins in a non-membranous environment3,15,19,26. This is the case of the nuclear receptor steroidogenic factor, which transcriptional control function is regulated by PI(3,4,5)P319, and poly-A polymerase which enzymatic activity is regulated by nuclear PI(4,5)P226. A regulatory role for IPs and PIs has been shown in many organisms including yeast22,27, mammalian cells19,23, Drosophila10 and worms28. Of significance is the role of these metabolites in trypanosomes, which diverged early from the eukaryotic lineage. These metabolites play an essential role in Trypanosoma brucei transcriptional control1,3, development8, organelle biogenesis and protein traffic29,30,31,32, and are also involved in controlling development and infection in the pathogens T. cruzi33,34,35, Toxoplasma36 and Plasmodium5,37. Hence, understanding the role of IPs and PIs in trypanosomes may help to elucidate new biological function for these molecules and to identify novel drug targets.
The specificity of protein and IP or PI binding depends on protein interacting domains and the phosphorylation state of the inositol13,38, although interactions with the lipid part of PIs also occurs19. The variety of IPs and PIs and their modifying kinases and phosphatases provides a flexible cellular mechanism for controlling protein function which is influenced by metabolite availability and abundance, the phosphorylation state of the inositol, and protein affinity of interaction1,3,13,38. Although some protein domains are well-characterized39,40,41, e.g., pleckstrin homology domain42 and SPX (SYG1/Pho81/XPR1) domains43,44,45, some proteins interact with IPs or PIs by mechanisms that remain unknown. For example, the repressor-activator protein 1 (RAP1) of T. brucei lacks canonical PI-binding domains but interacts with PI(3,4,5)P3 and control transcription of genes involved in antigenic variation3. Affinity chromatography and mass spectrometry analysis of IP or PI interacting proteins from trypanosome, yeast, or mammalian cells identified several proteins without known IP- or PI-binding domains8,46,47. The data suggest additional uncharacterized protein domains that bind to these metabolites. Hence, the identification of proteins that interact with IPs or PIs may reveal novel mechanisms of protein-metabolite interaction and new cellular regulatory functions for these small molecules.
The method described here employs affinity chromatography coupled to Western blotting or mass spectrometry to identify proteins that bind to IPs or PIs. It uses biotinylated IPs or PIs that are either cross-linked to streptavidin conjugated to agarose beads or alternatively, captured via streptavidin-conjugated magnetic beads (Figure 1). The method provides a simple workflow that is sensitive, non-radioactive, liposome-free and is suitable for detecting the binding of proteins from cell lysates or purified proteins3 (Figure 2). The method can be used in label-free8,46 or coupled to amino acid-labelled quantitative mass spectrometry47 to identify IP or PI-binding proteins from complex biological samples. Hence, this method is an alternative to the few methods available to study the interaction of IPs or PIs with cellular proteins and will help in understanding the regulatory function of these metabolites in trypanosomes and perhaps other eukaryotes.

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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Acetone</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">650501</td>
			<td style="width:377px;">Ketone</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Acetonitrile</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td>271004</td>
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		<tr height="25">
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Centrifuge Avanti J6-MI</td>
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			<td style="width:119px;">B1408</td>
			<td>Bottles for centrifugation of 1L of culture</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Control Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B000-1ml</td>
			<td>Affinity chromatography reagent - control</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">D-(+)-Glucose</td>
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			<td>Sugar, Added in PBS to keep cells viable</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Dithiothreitol (DTT)&#160;</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">1610610</td>
			<td>Reducing agent</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Dynabeads M-270 Streptavidin</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:119px;">65305</td>
			<td>Streptavidin beads for binding to biotin ligands</td>
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		<tr height="25">
			<td height="25" style="height:25px;">EDTA-free Protease Inhibitor Cocktail</td>
			<td style="width:163px;">Roche</td>
			<td style="width:119px;">11836170001</td>
			<td>Protease inhibitors</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Electrophoresis running buffer</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">1610732</td>
			<td style="width:377px;">25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Falcon 15 mL Conical Centrifuge Tubes</td>
			<td style="width:163px;">Corning Life Sciences</td>
			<td style="width:119px;">430052</td>
			<td>To centrifuge 10 mL cultures</td>
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			<td height="25" style="height:25px;width:323px;">Formic acid</td>
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			<td style="width:119px;">106526</td>
			<td>Acid</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Glycine</td>
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			<td style="width:119px;">G7126</td>
			<td>Amino acid</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">HMI-9 cell culture medium</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:119px;">ME110145P1</td>
			<td>Cell culture medium for T. brucei bloodstream forms</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Imperial Protein Stain</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:119px;">24615</td>
			<td>Coomassie staining for protein detection in SDS/PAGE</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Ins(1,4,5)P3 Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">Q-B0145-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
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			<td height="25" style="height:25px;width:323px;">Instant&#160;Nonfat&#160;Dry&#160;Milk</td>
			<td style="width:163px;">Thomas Scientific</td>
			<td style="width:119px;">C837M64</td>
			<td>Blocking reagent for Western blotting</td>
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			<td height="25" style="height:25px;width:323px;">Iodoacetamide</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">I6125</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Lab&#160;Rotator</td>
			<td style="width:163px;">Thomas Scientific</td>
			<td style="width:119px;">1159Z92</td>
			<td>For binding assays</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">LoBind Microcentrifuge Tubes</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:119px;">13-698-793</td>
			<td>Low protein binding tubes for mass spectrometry</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Nonidet&#160;P-40 (Igepal CA-630)</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">21-3277</td>
			<td>Detergent</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PBS, pH 7.4</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10010031</td>
			<td>Physiological buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Peroxidase substrate for chemiluminescence</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:119px;">32106</td>
			<td>Substrate for Western bloting detection of proteins</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PhosSTOP Phosphatase Inhibitor Cocktail Tablets</td>
			<td style="width:163px;">Roche</td>
			<td style="width:119px;">4906845001</td>
			<td>Phosphatase inhibitors</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(3)P PIP Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B003a-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(3,4)P2 PIP Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B034a-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(3,4,5)P3 diC8</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-3908-1mg</td>
			<td>Affinity chromatography reagent&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(3,4,5)P3 PIP Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B345a-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(3,5)P2 PIP Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B035a-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(4)P PIP Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B004a-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(4,5)P2 diC8</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-4508-1mg</td>
			<td>Affinity chromatography reagent&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(4,5)P2 PIP Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B045a-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(5)P PIP Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B005a-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Ponceau S solution</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7170</td>
			<td>Protein staining (0.1% [w/v] in 5% acetic acid)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Potassium hexacyanoferrate(III)</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">702587</td>
			<td>Potassium salt&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PtdIns PIP Beads</td>
			<td style="width:163px;">Echelon</td>
			<td style="width:119px;">P-B001-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PVDF Membrane</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">1620177</td>
			<td>For Western blotting&#160;</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Refrigerated centrifuge</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:119px;">5910 R</td>
			<td>Microcentrifuge for small volumes (e.g., 1.5 mL)</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Sodium dodecyl sulfate</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">862010</td>
			<td>Detergent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Sodium thiosulfate</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">72049</td>
			<td>Chemical&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">SpeedVac Vacuum Concentrators</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:119px;">SPD120-115</td>
			<td>Sample concentration (e.g., for mass spectrometry)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">T175 flasks for cell culture&#160;</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:119px;">159910</td>
			<td>To grow 50 mL T. brucei culture</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Trypsin, Mass Spectrometry Grade</td>
			<td style="width:163px;">Promega</td>
			<td style="width:119px;">V5280</td>
			<td>Trypsin for protein digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Urea</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">U5128</td>
			<td>Denaturing reagent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Vortex</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">02-215-418</td>
			<td>For mixing reactions</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Western blotting transfer buffer</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">1610734</td>
			<td>25 mM Tris, 192 mM glycine, pH 8.3 with 20% methanol</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Whatman&#160;3 mm paper</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">WHA3030861</td>
			<td>Paper for Wester transfer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">2-mercaptoethanol (14.2 M)</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">1610710</td>
			<td>Reducing agent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">2x Laemmli Sample Buffer</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">161-0737</td>
			<td>Protein loading buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">4&#8211;20% Mini-PROTEAN&#160;TGX Precast Protein Gels</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">4561094</td>
			<td>Gel for protein electrophoresis</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">4x Laemmli Sample Buffer</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">161-0747</td>
			<td>Protein loading buffer</td>
		</tr>
	</tbody>
</table>

identification of inositol phosphate or phosphoinositide interacting proteins by affinity chromatography coupled to western blot or mass spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

Analysis of RAP1 and PI(3,4,5)P3 interaction by affinity chromatography and Western blotting 
This example illustrates the application of this method to analyze the binding of PIs by RAP1 from T. brucei lysate or by recombinant T. brucei RAP1 protein. Lysates of T. brucei bloodstream forms that express hemagglutinin (HA)-tagged RAP1 were used in binding assays. RAP1 is a protein involved in transcriptional control of variant surface glycoprotein (VSG) genes<sup class="xref...

Watch this Scientific Journal Video about Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry at JoVE.

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Inositol phosphate and phosphoinositides are metabolites that have several regulatory functions in eukaryotes, including the control of gene expression, protein trafficking, signal transduction, and cell development. They perform these regulatory function by binding to proteins, thereby changing protein conformation, catalytic activity, or protein interactions. The identification of proteins that bind to inositol phosphate or phosphoinositides is essential to understand how those metabolites perform their regulatory function.This protocol has a simple workflow that's sensitive, non-radioactive, lyposome free and uses reagents that are commercially available. In this method, a finished chromatography with biotinylated inositol phosphate or phosphoinositides, is used to isolate interacting proteins, they are then identified by western blot, or mass spectrometry. For blood stream forms, grow T.brucei cells to mid log phase in HMI-9 media, supplemented with 10%FBS, at 37 degree Celsius, with 5%Carbon Dioxide.Keep the cell density between 800, 000 and 1.6 million cells per milliliter. When ready, centrifuge the cells at 1600 times G, and at room temperature for 10 minutes. Discard the supernatant, and gently resuspend the pellet in 10 milliletres of PBS-G pre-heated at 37 degrees Celsius to wash the cells.Centrifuge the cells at 1600 times G and at room temperature for five minutes to complete the wash. Repeat this washing procedure twice more. Then resuspend the pellet in one milliliter of PBS-G.Transfer this suspension to a one point five milliliter tube, then centrifuge at 1600 times G for five minutes. Discard the supernatant, and resuspend the pellet in zero point five milliliters of lysis buffer, supplemented with one point five X protease inhibitor cocktail, and one X phosphatase inhibitor cocktail that has been pre-chilled on ice to lyse the cells. Centrifuge the lysate at 1400 times G and at four degrees Celsius for 10 minutes.After this collect the supernatant, which contains the extracted parasite proteins, into a new one point five milliliter tube for the binding assays. Set aside 5%of the total lysate for western blot analysis. Collect 50 microliters of inositol phosphates or phosphoinositides conjugated to agarose beads, and add them 500 milliliters of binding buffer.Centrifuge at 1000 times G for one minute. Discard the supernatant, and resuspend in 50 microliters of binding buffer to equilibrate the beads. Use non-conjugated beads as a control, and use beads with different phosphate configurations including non-phosphorylated forms, to control for unspecific interactions.Next, add 50 microliters of IP or PI beads to the cell lysate or the purified proteins. Keep the volume of beads within 10%of the total lysate. If necessary, use binding buffer to adjust the volume of the binding reaction.Incubate the reaction for either one hour or overnight at four degrees Celsius, while rotating at 50rpm. After this, centrifuge the mix at 1000 times G, and at four degrees Celsius for one minute. Remove the supernatant, and keep the pellet, making sure to keep 5%of the supernatant for western blot analysis.Then add one milliliter of washing buffer and tap or swirl the tube to resuspend the resin. Centrifuge the reaction at 1000 times G, and at four degrees Celsius for one minute, and discard the supernatant. Repeat this process for a total of five washes.After this, add 50 microliters of 2X Laemmli buffer supplemented with 710 millimolar 2-mercaptoethanol to the beads. Tap or vortex to mix, and dilute the proteins. Next, heat at 95 degrees Celsius for five minutes, and centrifuge at 10, 000 times G for one minute.Collect the supernatant, and either freeze the eluate at minus 80 degrees Celsius, or proceed to western blot analysis. The method presented here is used to analyze the binding of PIs by repressor-activator protein 1 from T.brucei lysate, or by recombinant T.brucei repressor-activator protein 1 protein. As RAP1 lacks canonical PI binding domains, the binding assays are performed with PIs that are non-phosphorylated, or that are phosphorylated at different positions of the inositol ring, and with non-conjugated agarose beads.Western analysis shows that RAP1 binds preferentially to PI(3, 4, 5)P3 beads, but that it also binds to lesser extent to PI(4, 5)P2 beads. However, it did not bind to any other PIs or agarose beads. To test whether RAP1 binds directly to PIs, a C terminally tagged 6XHis recombinant RAP1 protein is expressed and purified to homogeneity from E.Coli.Western blotting shows that increasing the concentration of PI(3, 4, 5)P3, but not PI(4, 5)P2, inhibits the interaction of recombinant RAP1 with PI(3, 4, 5)P3 beads. The addition of T.brucei purified PIP5Pase enzyme to the reaction restored PI(3, 4, 5)P3 binding by recombinant RAP1. This is due to PIP5Pase dephosphorylation of free PI(3, 4, 5)P3, and thus indicates that the phosphorylation pattern of this metabolite is essential for our RAP1 binding.T.brucei proteins that bind to Ins(1, 4, 5)P3 are then identified by affinity chromatography followed by mass spectrometry. SDS-PAGE analysis shows enrichment in proteins eluted from Ins(1, 4, 5)P3 beads, compared to proteins eluted from the control agarose beads. Mass spectrometry analysis of the eluted proteins identified over 250 proteins of which 84 were enriched with Ins(1, 4, 5)P3 beads, compared to control beads.The enrichment of proteins bound to Ins(1, 4, 5)P3, compared to control beads, correlates with the protein signal detected by SDS-PAGE. A critical step in this protocol is the use of appropriate controls, to discriminate specific from non-specific interactions. We recommend using non-conjugated agarose beads, and beads conjugated with inositol phosphate or phosphoinositides, using various phosphate configurations, including non-phosphorylated forms.This method can be applied to other single-celled protozoan parasites, such as Trypanosoma cruzi, leishmania, or plasmodium. It can also be easily adapted to other organisms, including yeast or mammalian cells. This method can be coupled to quantitative mass spectrometry, such as SILAC, to identify dynamic interactions of proteins with inositol phosphate or phosphoinostides.In can also be combined with subcellular fractionation, to identify organelle specific proteins that bind to these metabolites. This approach has helped to identify proteins that bind to inositol phosphate and phosphoinositides in T.brucei, mammalian cells, and yeast. It has helped identify new protein domains that bind to phosphoinositides.It has paved the way to understand the regulatory function of these metabolites, their role in gene expression, cell development, and signal transduction in eukaryotes. Some of the reagents in this protocol are toxic, or flammable. Use personal protective equipment, and where necessary, work in a fume hood.

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8.3K 조회수.  McGill University. 이노시톨 인산염 및 인산조류는 진핵생물에 여러 가지 조절 기능이 있는 대사산물로, 유전자 발현, 단백질 인신매매, 신호 트랜스듀션 및 세포 발달의 조절을 포함한다. 그(것)들은 단백질에 결합하여, 그로 단백질 형성, 촉매 활동, 또는 단백질 상호 작용을 변화시킴으로써 이 규정 기능을 능력을 발휘합니다. 이노 시 톨 인산염 또는 인포이 노 시 티드에 바인딩 하는 단백질?...

비디오: Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry