Lipids

, Volume 35, Issue 11, pp 1289–1297

Phospholipid identification and quantification of membrane vesicle subfractions by 31P−1H two-dimensional nuclear magnetic resonance

  • B. Larijani
  • D. L. Poccia
  • L. Charles Dickinson
Method

DOI: 10.1007/s11745-000-0645-4

Cite this article as:
Larijani, B., Poccia, D.L. & Dickinson, L.C. Lipids (2000) 35: 1289. doi:10.1007/s11745-000-0645-4

Abstract

An approach to the direct quantification of phospholipids from two-dimensional 31P−1H nuclear magnetic resonance (NMR) spectroscopy with isotropic proton mixing has been developed as a general method for phospholipid analysis of minor membrane vesicle subfractions. Membrane vesicles were subfractionated by sedimentation to density equilibrium in a sucrose gradient, and a modified Folch method was employed to extract their phospholipids. The coefficient for the NMR detection efficiency of each phospholipid and the relative mole percentage of the phospholipids present in the membrane vesicles were calculated. We demonstrate low detection limits such that relative concentrations of phospholipids in membrane subfractions may be determined even in the submicromolar range.

Abbreviations

AAPC

β-acetyl-γ-O-alkyl-l-α-phosphatidylcholine

1D

one-dimensional

2D

two-dimensional

DPPC

l-α-phosphatidylcholine, dipalmitoyl

DPPE

dl-α-phosphatidylethanolamine, dipalmitoyl

DPPS

dl-α-phosphatidyl-l-serine, dipalmitoyl

DPSM

dipalmitoyl sphingomyelin

ER

endoplasmic reticulum

GARP

globally optimized alternating phase rectangular pulse decoupling

HMQC

heteronuclear multiple quantum coherence

HMSQ

heteronuclear single quantum coherence

K

coefficient of efficiency of NMR detection

LB

lysis buffer

LPC

lysophosphatidylcholine

MLEV

Malcolm Levitt decoupling scheme

MV

membrane vesicle

MWB

membrane wash buffer

NMR

nuclear magnetic responance

PE

phosphatidylethanolamine

PI

l-α-phosphatidylinositol sodium salt

PMSF

phenylmethylsulfonylfluoride

TN

Tris-NaCl

TOCSY

total correlation spectroscopy

Copyright information

© AOCS Press 2000

Authors and Affiliations

  • B. Larijani
    • 1
  • D. L. Poccia
    • 1
  • L. Charles Dickinson
    • 2
  1. 1.Department of BiologyAmherst CollegeAmherst
  2. 2.Conte National Center for Polymer Research, Department of Polymer Science and EngineeringUniversity of Massachusetts at AmherstAmherst
  3. 3.Imperial Cancer Research FundLondonUK

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