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;

The author has nothing to disclose.

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.

<table><tbody><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>650501</td><td>Ketone</td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>271004</td><td>Solvent&amp;nbsp;</td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich</td><td>A6141</td><td>Inorganic salt</td></tr><tr><td>Centrifuge Avanti J6-MI</td><td>Beckman Coulter</td><td>Avanti J6-MI</td><td>Centrifuge for large volumes (e.g., 1L)</td></tr><tr><td>Centrifuge botles</td><td>Sigma-Aldrich</td><td>B1408</td><td>Bottles for centrifugation of 1L of culture</td></tr><tr><td>Control Beads</td><td>Echelon</td><td>P-B000-1ml</td><td>Affinity chromatography reagent - control</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>Sugar, Added in PBS to keep cells viable</td></tr><tr><td>Dithiothreitol (DTT)&amp;nbsp;</td><td>Bio-Rad</td><td>1610610</td><td>Reducing agent</td></tr><tr><td>Dynabeads M-270 Streptavidin</td><td>ThermoFisher Scientific</td><td>65305</td><td>Streptavidin beads for binding to biotin ligands</td></tr><tr><td>EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11836170001</td><td>Protease inhibitors</td></tr><tr><td>Electrophoresis running buffer</td><td>Bio-Rad</td><td>1610732</td><td>25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3</td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes</td><td>Corning Life Sciences</td><td>430052</td><td>To centrifuge 10 mL cultures</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>106526</td><td>Acid</td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td>Amino acid</td></tr><tr><td>HMI-9 cell culture medium</td><td>ThermoFisher Scientific</td><td>ME110145P1</td><td>Cell culture medium for &lt;em&gt;T. brucei&lt;/em&gt; bloodstream forms</td></tr><tr><td>Imperial Protein Stain</td><td>ThermoFisher Scientific</td><td>24615</td><td>Coomassie staining for protein detection in SDS/PAGE</td></tr><tr><td>Ins(1,4,5)P3 Beads</td><td>Echelon</td><td>Q-B0145-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>Instant&amp;nbsp;Nonfat&amp;nbsp;Dry&amp;nbsp;Milk</td><td>Thomas Scientific</td><td>C837M64</td><td>Blocking reagent for Western blotting</td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I6125</td><td>Alkylating reagent for cysteine proteins or peptides</td></tr><tr><td>Lab&amp;nbsp;Rotator</td><td>Thomas Scientific</td><td>1159Z92</td><td>For binding assays</td></tr><tr><td>LoBind Microcentrifuge Tubes</td><td>ThermoFisher Scientific</td><td>13-698-793</td><td>Low protein binding tubes for mass spectrometry</td></tr><tr><td>Nonidet&amp;nbsp;P-40 (Igepal CA-630)</td><td>Sigma-Aldrich</td><td>21-3277</td><td>Detergent</td></tr><tr><td>PBS, pH 7.4</td><td>ThermoFisher Scientific</td><td>10010031</td><td>Physiological buffer</td></tr><tr><td>Peroxidase substrate for chemiluminescence</td><td>ThermoFisher Scientific</td><td>32106</td><td>Substrate for Western bloting detection of proteins</td></tr><tr><td>PhosSTOP Phosphatase Inhibitor Cocktail Tablets</td><td>Roche</td><td>4906845001</td><td>Phosphatase inhibitors</td></tr><tr><td>PI(3)P PIP Beads</td><td>Echelon</td><td>P-B003a-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PI(3,4)P2 PIP Beads</td><td>Echelon</td><td>P-B034a-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PI(3,4,5)P3 diC8</td><td>Echelon</td><td>P-3908-1mg</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PI(3,4,5)P3 PIP Beads</td><td>Echelon</td><td>P-B345a-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PI(3,5)P2 PIP Beads</td><td>Echelon</td><td>P-B035a-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PI(4)P PIP Beads</td><td>Echelon</td><td>P-B004a-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PI(4,5)P2 diC8</td><td>Echelon</td><td>P-4508-1mg</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PI(4,5)P2 PIP Beads</td><td>Echelon</td><td>P-B045a-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PI(5)P PIP Beads</td><td>Echelon</td><td>P-B005a-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>Ponceau S solution</td><td>Sigma-Aldrich</td><td>P7170</td><td>Protein staining (0.1% [w/v] in 5% acetic acid)</td></tr><tr><td>Potassium hexacyanoferrate(III)</td><td>Sigma-Aldrich</td><td>702587</td><td>Potassium salt&amp;nbsp;</td></tr><tr><td>PtdIns PIP Beads</td><td>Echelon</td><td>P-B001-1ml</td><td>Affinity chromatography reagent&amp;nbsp;</td></tr><tr><td>PVDF Membrane</td><td>Bio-Rad</td><td>1620177</td><td>For Western blotting&amp;nbsp;</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5910 R</td><td>Microcentrifuge for small volumes (e.g., 1.5 mL)</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>862010</td><td>Detergent</td></tr><tr><td>Sodium thiosulfate</td><td>Sigma-Aldrich</td><td>72049</td><td>Chemical&amp;nbsp;</td></tr><tr><td>SpeedVac Vacuum Concentrators</td><td>ThermoFisher Scientific</td><td>SPD120-115</td><td>Sample concentration (e.g., for mass spectrometry)</td></tr><tr><td>T175 flasks for cell culture&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>159910</td><td>To grow 50 mL &lt;em&gt;T. brucei&lt;/em&gt; culture</td></tr><tr><td>Trypsin, Mass Spectrometry Grade</td><td>Promega</td><td>V5280</td><td>Trypsin for protein digestion</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5128</td><td>Denaturing reagent</td></tr><tr><td>Vortex</td><td>Fisher Scientific</td><td>02-215-418</td><td>For mixing reactions</td></tr><tr><td>Western blotting transfer buffer</td><td>Bio-Rad</td><td>1610734</td><td>25 mM Tris, 192 mM glycine, pH 8.3 with 20% methanol</td></tr><tr><td>Whatman&amp;nbsp;3 mm paper</td><td>Sigma-Aldrich</td><td>WHA3030861</td><td>Paper for Wester transfer</td></tr><tr><td>2-mercaptoethanol (14.2 M)</td><td>Bio-Rad</td><td>1610710</td><td>Reducing agent</td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>161-0737</td><td>Protein loading buffer</td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN&amp;nbsp;TGX Precast Protein Gels</td><td>Bio-Rad</td><td>4561094</td><td>Gel for protein electrophoresis</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>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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8.4K Views.  McGill University. 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.

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

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Chemistry

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.

A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...