Selected Experiments

Mass Spectrometer Facility
Department of Biochemistry
Wake Forest University School of Medicine
391 Technology Way
Building A1, Room 340
Winston-Salem, NC 27101

Relative Intensity

Phospholipids are the major lipid component of cell membranes. These molecules are composed of hydrophobic fatty acids, glycerol, and phosphate. The phosphate is usually attached to a polar organic residue like choline, ethanolamine, serine, inositol, etc.

Analysis of phospholipids in lipid extracts of tissues, plasma, or cell culture.

The preceding figure shows a typical positive parent ion spectrum of a lipid extract with detection at 184 m/z to identify choline-containing phospholipids. The total number of carbon atoms in the fatty acid components of the diacylphosphatidylcholines is given at the top of the figure. Typical fatty acid combinations are listed above the peak clusters. 1-O-alkyl-2-acylphosphatidylcholines have masses that are 14 Da lower than diacylphosphatidylcholines carrying similar carbon chains at the sn-1 and sn-2 positions on the glycerol backbone.

Lipid References:

1. DeLong, C. J., et al.: Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography. J. Lipid Res., 42:1959-68 (2001).
2. Lee, J-Y., et al.: Pre-b high density lipoprotein (HDL) has two metabolic fates in human apolipoprotein A-I transgenic mice. J. Lipid Res., 45:716-728 (2004).
3. Edwards, I. J., et al.: Differential effects of delivery of n-3 fatty acids to human cancer cells by low density lipoproteins versus albumin. Clin. Cancer Res., 10:8275-83 (2004).
4. Owen, J.S., et al.: An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry. J. Lipid Res., 46:373-82 (2005).
5. Tsui, Z-C., et al.: Profiling gangliosides in biological samples using electrospray-tandem mass spectrometry. Anal. Biochem., 341:251-8 (2005).
Mass spectrometry is a tool that can provide the molecular weight of an intact protein and after digesting the protein by a protease can give detailed information about the primary structure and post-translational modifications to the protein. The Q-TOF has been used to study the structure of apolipoprotein A-I (apoA-I) bound to recombinant phospholipid disks. The first step in the analysis is to react lysines with lysine-reactive cross-linking reagents and to then digest the cross-linked protein after purification by SDS-PAGE. These studies have suggested that two molecules of apoA-I are wrapped around the periphery of the lipid disk in an antiparallel orientation. The N-and C-terminal regions appear to interact and bend back on themselves (Bhat, S.,: Inter-molecular contact between globular N-terminal fold and C-terminal domain of ApoA-I stabilize its lipid-bound conformation: Studies employing chemical cross-linking and mass spectrometry.  J. Biol. Chem., 280:33015-33025 (2005)). These studies emphasized the need to have both highly accurate mass determinations and the sequence data for unambiguously identifying cross-linked peptides. A typical MS/MS spectrum is shown below for two peptides connected across lysines using dithiobis(succinimidylpropionate).

QKVEPLR LHELQEKSPLGEEMR

Protein References:

1. Li, H-H., et al.: Apo A-I structure on discs and spheres: helix registry and conformational states. J. Biol. Chem., 277:39093-39101 (2002).
2. Alexander, E.T., et al.: ApolipoproteinA-I helix 6 negatively charged residues attenuate LCAT reactivity. Biochemistry, 44:5409-5419 (2005).
3. Poole, L.B., et al.: Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjug. Chem., 16:1624-1628 (2005).