AfCS > AfCS laboratories > Lipidomics Laboratory

ALLIANCE FOR CELLULAR SIGNALING

LIPIDOMICS LABORATORY

Vanderbilt University Medical Center
Nashville, Tennessee

Overview

The primary goal of the Alliance for Cellular Signaling (AfCS) Lipidomics Laboratory is to achieve comprehensive analysis of membrane lipid composition in response to cell surface receptor stimulation. Changes are identified in cellular glycerophospholipid (GPL) composition as a consequence of activating signaling pathways in the macrophage model cell line RAW 264.7. Just as identifying the temporal sequence of changes in gene transcription and proteomic patterns has revolutionized biology, obtaining a comprehensive picture of changes in membrane lipid composition will allow us to understand the biophysical and biochemical processes that mediate membrane signaling pathways. These processes influence cell shape and bilayer fluidity, which in turn modulate cellular function. In this way, the molecular events in the cellular membrane that follow activation of G protein-coupled receptors (GPCRs) on the cell surface will be revealed.

As a complete picture of changes in global membrane lipid composition emerges, we will integrate our findings with those of other AfCS projects to determine which specific lipid species may have roles in the temporal and spatial signaling events being studied (e.g., imaging the translocation of proteins containing PIP3-binding domains and gene transcription). In the absence of such detailed information, we do not know whether pattern changes in "housekeeping" GPLs (e.g., phosphatidylserine [PS], phosphatidylethanolamine [PE], or phosphatidylcholine [PC]), which form the structural scaffold of the cell bilayer, have unappreciated roles in complex biological processes. Obtaining detailed lipid array analysis will allow us to formulate a working model as to how such dynamic changes in membranes influence complex biological processes, such as chemotaxis.

Mass spectra are currently being acquired on a Finnigan Quantum TSQ with on-line high performance liquid chromatography (HPLC). All samples are electrosprayed in both positive and negative ion mode. The effective mass range is m/z 500-1200 (negative mode) and 400-1200 (positive mode). Typically, less than 6 x 10⁶ cell equivalents of lipid extract are being used to obtain complete spectra. The relative responses for phospholipids are more dependent on the chemistry of the head group than the fatty acyl composition within a phospholipid class, which allows estimation of the relative changes in lipid composition using standard mixtures of a member of each class. The relative sensitivities of detection of different phospholipid classes are determined using a mixture of equimolar PC, PE, sphingomyelin (SM), lysophosphatidylcholine (LPC), and diacylglycerol (DAG) standards for the positive ion mode and a mixture of phosphatidylinositol (PI), PE, PS, phosphatidic acid (PA), and phosphatidylglycerol (PG) for the negative ion mode. PE intensities observed in both ion modes are used to normalize the spectra with respect to each other. Due to the mass accuracy and resolution of instrumentation, we are able to determine relative changes of specific lipid species as a function of global lipid composition.

Staff

Alliance for Cellular Signaling
Lipidomics Laboratory
412B Preston Research Building
23^rd Ave. South & Pierce
Nashville, Tennessee 37232-6600
Phone: 615-936-3888
Fax: 615-343-6532

Director	H. Alex Brown, Ph.D.
Project Coordinator	Jeffrey Forrester, Ph.D.
Project Scientist	Stephen Milne, Ph.D.
Project Scientist	Pavlina Ivanova, Ph.D.
Lab Manager	Michelle Armstrong
Data Analyst	Mark Byrne
Technical Staff	Andrew Goodman

Slide Presentation from May 23-26, 2004, AfCS Annual Meeting

• "Lipids", Alex Brown

Goals for Year 5 (9/04-8/05)

• Complete the single and dual ligand screens as determined by the AfCS Steering Committee.
• Begin the outlined perturbation studies for the larger ligand screen list (initially piloted for the FXM ligands), including the use of small molecule inhibitors and siRNAs to disrupt targeted nodes in the signaling pathways of interest.
• Develop a new direct infusion procedure for lipidomic analysis of diacylglycerol species.
• Expand on the analysis of poly phosphoinositides in RAW264.7 cells. Recent advances have permitted an identification of a broad number of species. In the coming year we will be able to resolve the positions of the poly phosphates on the inositol ring (D1-D6). This will be accomplished through a systematic combination of LC-MS procedures and selective enzymatic elimination of peaks (e.g., use of PLC to discriminate PI3,4P2 from PI4,5P2).