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

Summary

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.

Abstract

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.

Introduction

Phosphoinositides (PtdInsPs) are important signaling phospholipids that help regulate a variety of cellular functions, including signal transduction, membrane trafficking and gene expression, which then modulate higher-order cell behavior such as cell division, organelle identity and metabolic activity^1-3. There are seven species of PtdInsPs that are derived from the phosphorylation of the 3, 4, and/or 5 positions of the inositol head group of phosphatidylinositol (PtdIns), the parent phospholipid. Importantly, the seven PtdInsPs are unequally distributed and the local concentration of each PtdInsP species can increase or decrease at specific subcellular si....

Protocol

Note: The text below describes in detail a method to measure PtdInsPs in yeast. It provides the experimental details for labeling yeast cells with ³H-myo-inositol, extracting and deacylating lipids and an HPLC-elution protocol to fractionate and quantify deacylated PtdInsPs. Please note that labeling, deacylation, resolution and quantification of PtdInsPs in mammalian cells require optimization and longer HPLC-elution profiles. These details can be found elsewhere, though we discuss some of aspects in the Discussion. Overall, the methodology to extract lipids, deacylate and extract the water-soluble Gro-InsPs from yeast is given and is illustrated ....

Results

Using this method, yeast PtdInsPs were metabolically labelled with ³H-myo-inositol. After labeling, the phospholipids were precipitated with perchloric acid, followed by phospholipid deacylation and extraction of the water-soluble Gro-InsPs (Figure 1). At this stage, it is important to quantify the total radioactive signal associated with the extracted Gro-InsPs by liquid scintillation to ensure sufficient signal-to-noise ratio for the very low-abundan.......

Discussion

This article details the experimental protocol required to quantify cellular levels of PtdnsPs by HPLC-coupled flow scintillation from yeast. The methodology enables the metabolic labeling of PtdInsPs with ³H-myo-inositol, followed by lipid processing and extraction of water-soluble ³H-Gro-InsPs, HPLC fractionation and analysis. Using this method, the relative levels of PtdInsPs in cells under various conditions can be quantified, as is shown for PtdIns(3,5)P₂ in wild-type, v.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-Butanol	Biobasic	BC1800	Reagent grade
Ammonium phosphate dibasic	Bioshop	APD001	ACS grade
Ammonium sulfate	Biobasic	ADB0060	Ultra Pure grade
Autosampler	Agilent	G1329B	Agilent 1260 infinity series
Biotin	Sigma	B4501
Boric acid	Biobasic	BB0044	Molecular biology grade
Calcium Chloride	Biobasic	CT1330	Ahydrous, industrial grade
Calcium pantothenate	Sigma	C8731
Copper(II) sulfate	Sigma	451657	Anhydrous
D-Glucose	Biobasic	GB0219	Anhydrous, biotech grade
Dulbecco's modification of Eagle's Medium	Life	11995-065	With 4.5 g/L glucose, 110 mg/L pyruate, L-glutamine
Dulbecco's modification of Eagle's Medium	MP biomedicals	0916429	With 4.5 g/L glucose, without L-gluatmine, without inositol
EDTA	Biobasic	EB0107	Acid free, ultra pure grade
Ethyl ether	Caledon labs	1/10/4700	Anhydrous, reagent grade
Ethyl formate	Sigma	112682	Reagent grade
Fetal bovine serum	Wisent	080-450	US origin, premium quality, heat inactivated
Fetal Bovine Serum, Dialyzed	Life	26400044	US origin
FlowLogic U	LabLogic Systems Ltd	SG-BXX-05	Scintillation fluid for flow scintillation
Folic acid	Biobasic	FB0466	USP grade
HEPES buffer solution	Life	15630080	1 M solution
Inositol, Myo-[2-3H(N)]	Perkin Elmer	NET114005MC	9:1 ethanol to water
Insulin-Transferrin-Selenium-Ethanolamine	Life	51500056	100x solution
Iron(III) chloride	Sigma	157740	Reagent grade
Laura - Chromatography data collection and analysis software	LabLogic Systems Ltd	Version 4.2.1.18	Flow scintillator software
L-glutamine	Sigma	G7513	200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
Magnesium Chloride	Sigma	M8266	Anhydrous
Manganese sulfate	Biobasic	MB0334	Monohydrate, ACS grade
Methanol	Caledon labs	6701-7-40	HPLC Grade
Methylamine solution	Sigma	426466	40% (v/v)
Monopotassium phosphate	Biobasic	PB0445	Anhydrous, ACS grade
Nicotinic acid	Biobasic	NB0660	Reagent grade
OpenLAB CSD ChemStation	Agilent	Rev. C.01.03	HPLC software
p-aminobenzoic acid (PABA)	Bioshop	PAB001.100	Free acid
Penicillin-Streptomycin	Sigma	P4333	100X, liquid, stabilized, sterile-filtered, cell culture tested
Perchloric acid	Sigma	244252	ACS reagent, 70%
PhenoSpher SAX column	Phenomenex	00G-315-E0	5 µm, 80 Å, 250 x 4.6 mm
Phosphoric acid	Caledon labs	1/29/8425	Reagent grade
Potassium Chloride	Biobasic	PB0440	ACS grade
Potassium iodide	Biobasic	PB0443	ACS grade
Pyridoxine hydrochloride	Sigma	P9755
Quaternary pump	Agilent	G1311C	Agilent 1260 infinity series
Riboflavin	Bioshop	RIB333.100	USP grade
Sodium Chloride	Biobasic	DB0483	Biotech grade
Sodium molybdate	Sigma	243655
Thermostatted Column Compartment	Agilent	G1316A	Agilent 1260 infinity series
Thiamine hydrochloride	Sigma	T4625	Reagent grade; make solution of 0.02% (w/v), forms a suspension. mix and freeze aliquots
Ultima Gold	Perkin Elmer	6013321	Scintillation coctail for liquid scintillation counting
Zinc sulfate	Biobasic	ZB2906	Heptahydrate, reagent grade
β-RAM 4	IN/US systems	Model 4	Flow scintillator - 500 µl flow cell; alternative Radiomatic Flow Scintillator Analyser by Perkin Elmer