Imaging of Exposed Headgroups and Tailgroups of Phospholipid Membranes by Mass Spectrometry

Cellular membrane function is driven by a complex set of physical and chemical properties including molecular density, spatial distribution, and molecular orientation in the leaflets of the phospholipid bilayer. Here we show, for the first time, by using secondary ion mass spectrometry (SIMS), that it is possible not only to determine the chemical identity of the membrane molecules but also to determine whether the headgroup or the tailgroup of the phospholipid molecule is exposed to the vacuum. This remarkable signature is demonstrated on both freezefractured, frozen-hydrated red blood cell membranes and on synthetic membranes made by Langmuir -Blodgett (LB) techniques. Our experiments were performed by means of imaging timeof-flight secondary ion mass spectrometry (TOF-SIMS). With this method the specimen surface is probed with a finely focused (200 nm) pulsed Ga + ion beam which desorbs ions from a well-defined area. 1 An image is created by rastering the ion beam over the surface and collecting a series of mass spectra at each point. The intensity of the ions as a function of position provides spatial information about the distribution of target molecules. Lipid samples were analyzed at temperatures of less than 170 K to prevent the sublimation of lipid in a vacuum. The behavior of the SIMS fragment ions from oriented organic films is best revealed using LB technology where the molecular configuration is precisely determined. We constructed oriented LB films of phosphatidylcholine dipalmitoyl (DPPC) such that either a headgroup or a tailgroup is forced toward the air -film interface. To prepare a film with the headgroup pointing away from the substrate surface, a C 16 alkane thiol monolayer on gold was inserted through a layer of DPPC on a water subphase. 2 Th thiol substrate is important because it presents a nonpolar surface that induces physisorption of the lipid tailgroups. To prepare a film with the tailgroup pointing away from the surface, a SiO 2/ Si substrate was pulled through a layer of DPPC on the same water subphase. This substrate presents a polar surface that preferentially binds the lipid headgroup. The LB films were made at a highly compressed region of the surface pressure/area isotherm at a constant pressure of 35 mN/m. Each type of sample was confirmed to be one monolayer in thickness using ellipsometry.3 Random or unoriented DPPC films were prepared by depositing a chloroform solution of DPPC onto a Si wafer under ambient conditions and allowing it to dry. The relevant portion of the positive ion TOF-SIMS spectra associated with the above preparations is shown in Figure 1. The fragment atm/z 184 corresponds to the well-known phosphocholine headgroup moiety, 4 while the fragments near m/z311 are associated with the alkyl tailgroup. From the spectra, it is clear that the intensity of specific ions correlates strongly with a specific molecular orientation. In the heads up configuration, m/z 184 is observed, while in the tails up configuration, the m/z 311 species predominates. The peak at m/z 311, previously unreported, has the molecular formula C 19H35O3 as determined from its exact mass. These results are consistent with predictions of molecular dynamics computer simulations. 5 Although there have been other reports which indicate the SIMS ion yield intensities depend on which functional groups are exposed to the ion beam, 6 we believe these results are the first to indicate that specific fragment ions are associated with specific configurations. It would be of special interest to utilize this mass spectral signature to probe the structure of intact biological membranes. To test this idea, we have examined the surface of rapidly frozen human red blood cells that have been freeze fractured directly in the vacuum environment of the mass spectrometer. These types of cells make an excellent model system since they have no internalized membranes and have been extensively characterized by many techniques. 7 We have employed freeze fracture to prepare these samples since previous studies have shown that this protocol is the most effective procedure for exposing an unperturbed membrane surface. 8