Name: identification of inositol phosphate or phosphoinositide interacting proteins by affinity chromatography coupled to western blot or mass spectrometry
Uploaded: 2019-07-26T13:30:00
Description: 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.

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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			<td height="25" style="height:25px;width:323px;">Acetone</td>
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			<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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			<td style="width:119px;">430052</td>
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			<td>Amino acid</td>
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			<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>
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			<td height="25" style="height:25px;width:323px;">Imperial Protein Stain</td>
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			<td style="width:119px;">24615</td>
			<td>Coomassie staining for protein detection in SDS/PAGE</td>
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			<td height="25" style="height:25px;width:323px;">Ins(1,4,5)P3 Beads</td>
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			<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>
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			<td style="width:119px;">C837M64</td>
			<td>Blocking reagent for Western blotting</td>
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			<td style="width:163px;">Thomas Scientific</td>
			<td style="width:119px;">1159Z92</td>
			<td>For binding assays</td>
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			<td height="25" style="height:25px;width:323px;">LoBind Microcentrifuge Tubes</td>
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			<td style="width:119px;">13-698-793</td>
			<td>Low protein binding tubes for mass spectrometry</td>
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			<td style="width:119px;">10010031</td>
			<td>Physiological buffer</td>
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			<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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			<td>Affinity chromatography reagent&#160;</td>
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			<td style="width:119px;">P-3908-1mg</td>
			<td>Affinity chromatography reagent&#160;</td>
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			<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>
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		<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>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">PI(4)P PIP Beads</td>
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			<td style="width:119px;">P-B004a-1ml</td>
			<td>Affinity chromatography reagent&#160;</td>
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		<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>
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		<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>
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		<tr height="25">
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			<td height="25" style="height:25px;width:323px;">Trypsin, Mass Spectrometry Grade</td>
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			<td height="25" style="height:25px;width:323px;">Vortex</td>
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			<td style="width:119px;">02-215-418</td>
			<td>For mixing reactions</td>
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		<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>
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			<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>
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		<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 ...

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Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...