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 height="25" style="height:25px;width:323px;">Dithiothreitol (DTT)&#160;</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:323px;">Dynabeads M-270 Streptavidin</td>
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			<td style="width:119px;">65305</td>
			<td>Streptavidin beads for binding to biotin ligands</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 height="25" style="height:25px;width:323px;">Falcon 15 mL Conical Centrifuge Tubes</td>
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			<td style="width:119px;">430052</td>
			<td>To centrifuge 10 mL cultures</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>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<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>
			<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>
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			<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>
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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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		<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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			<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 height="25" style="height:25px;width:323px;">PhosSTOP Phosphatase Inhibitor Cocktail Tablets</td>
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			<td>Phosphatase inhibitors</td>
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			<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>
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			<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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			<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>
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		<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>
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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>
			<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>
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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">
			<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>
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			<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>
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			<td height="25" style="height:25px;width:323px;">PtdIns PIP Beads</td>
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			<td>Affinity chromatography reagent&#160;</td>
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			<td style="width:119px;">1620177</td>
			<td>For Western blotting&#160;</td>
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			<td height="25" style="height:25px;width:323px;">Refrigerated centrifuge</td>
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			<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>
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			<td height="25" style="height:25px;width:323px;">Sodium thiosulfate</td>
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			<td style="width:119px;">72049</td>
			<td>Chemical&#160;</td>
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			<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>
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		<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>
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		<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>
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			<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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Unit 1

Intracellular Membrane Traffic

SCIENTISTS IN ACTION

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

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 biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

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

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

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 structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

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

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

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

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

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

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...