Mass Spectrometer

Selected Experiments

LIPIDS.  Glycerohospholipids are the major lipid component of cell membranes. They 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.

Relative Intensity

The preceding figure shows a typical positive precursor ion spectrum of a lipid extract to identify choline-containing glycerophospholipids (Delong et al 2001). The total number of carbon atoms in the fatty acid components of the diacylglycerophosphatidylcholines is given at the top of the figure. Typical fatty acid combinations are listed above the peak clusters. 1-O-alkyl-2-acylglycerophosphatidylcholines have masses that are 14 Da lower than the similar diacyl-glycerophosphatidylcholines carrying similar carbon chains at the sn-1 and sn-2 positions of the glycerol backbone. Our laboratory is particularly skilled at quantifying platelet activating factor (PAF) from a variety of cell sources.

In addition to glycerophosphocholine we analyze the other glycerophospholipid classes with MS/MS fatty acid determination, sphingomyelins, glycosylceramides, ceramides, sphingosine-1-phosphate, cholesteryl esters, triglycerides, etc.

Representative Lipid References:

1. 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).
2. Owen, J.S., et al.: An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry.  J. Lipid Res., 46:373-82 (2005).
3. Tsui, Z-C., et al.: Profiling gangliosides in biological samples using electrospray-tandem mass spectrometry.  Anal. Biochem., 341:251-8 (2005).
4. Hicks, A.M., et al.: Unique molecular signatures of glycerophospholipid species in different rat tissues analyzed by tandem mass spectrometry. Biochim. Biophys. Acta, 1761:1022-1029 (2006).
5. Witzenrath, M., et al.: Role of platelet-activating factor in pneumolysin-induced acute lung injury. Critical Care Medicine, 35:1756-1762 (2007).
6. Cui, Z. and Thomas, M.J.: Phospholipid profiling by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 877:2709-2715 (2009).
7. Wilhelm, A.J., et al.: Apolipoprotein A-I Modulates Regulatory T Cells in Autoimmune LDLr-/-, ApoA-I-/- Mice. J Biol Chem, 285:36158-36169 (2010).
8. Potteaux, S., et al.: Suppressed monocyte recruitment drives macrophage removal from atherosclerotic plaques of ApoE-/- mice during disease regression. J Clin Invest, 121:2025-2036 (2011).

PROTEINS. 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. For example, the Q-TOF has been used to study the structure of apolipoprotein A-I (apoA-I) bound to recombinant phospholipid disks. Accessible lysines react with lysine-reactive cross-linking reagents then digested with trypsin after SDS-PAGE separation. MS/MS analysis of the peptides suggested that two molecules of apoA-I are wrapped around the periphery of the lipid disk in an antiparallel orientation. Below is a typical MS/MS spectrum for two peptides connected across lysines using the cross-linking agent dithiobis(succinimidylpropionate).
The N-and C-terminal regions appear to interact and bend back on themselves (see structure below). These studies emphasized the need to have both highly accurate mass determinations and sequence data to unambiguously identifying cross-linked peptides. The following figures

Structure1  Structure2

show the conformation of apoA-I. The left panel shows the two apoA-Is as a ribbon cartoons in the antiparallel conformation with the cross-linked lysines appropriately spaced.  The right panel shows the same apoA-I conformation on a phospholipids disk with the apoA-I and palmitoyl-oleoylphosphatidylcholine displayed as atomic spheres.
    Expression of the L159R mutation in apoA-I, apoA-IFin, reduces the concentration of native plasma apoA-I. We have shown that mass spectrometry can be used measure the amount of mutant protein in a background of native protein. This analytical method has been demonstrated using 1 µL of mouse plasma in which the proteins were first separated by SDS-PAGE and then digested with trypsin. The figure below shows the lipid disks that carry apoA-I and the tryptic peptides that are characteristic of each protein.



 Work in the Poole lab was emphasized identifying sulfenic acid-forming proteins including the use of mass spectrometry to follow the addition of other labeling agents such as dimedone and 1,3-cyclohexanedione. Using the Waters Q-TOF we have identified a characteristic ion generated following fragmentation of fluorescein-labeled peptides, m/z= 576.13. As shown below, the appearance of this ion is used to trigger data dependent MS/MS sequencing of the peptide that yields the m/z= 576.13 ion. The goal of these studies is to conduct global analysis for the presence of cysteinesulfenic acids in whole cell digests where many other, unlabeled peptides are also present in the mixture. The sequencing is absolutely necessary to identify the protein that has a cysteine participating in redox cycling.




Sample Isolation new


Representative 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).
5. Poole, L.B., et al.: Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjug. Chem., 16:1624-1628 (2005).
3. Bhat, S., et al.: 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).
4. Owen, J.S., et al.: Ratio determination of plasma wild-type and mutant apoA-I using mass spectrometry quantification, purification and expression of L159R apoA-I (apoA-IFin). J. Lipid Res., 48:226-234 (2006).
5. Bhat, S., et al.: Conformational adaptation of apolipoproteinA-I to discretely sized phospholipid complexes. Biochemistry, 46:7811-7821 (2007).
6. Poole, L.B., et al.: Fluorescent and affinity-based tools to detect cysteine sulfenic acid formation in proteins. Bioconjugate Chem, 18:2004–2017 (2007).
7. Thomas, M.J., et al.: Three-dimensional models of high density lipoprotein apoA-I: Implications for its assembly and function. J. Lipid Res., 49:1875-1883 (2008).
8. Bhat, S., et al.: Conformation of Dimeric ApoA-IMilano on Recombinant Lipoprotein Particles. Biochemistry, 49:5213-5224 (2010).


Quick Reference

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