- Mass spectrometry offers insight into membrane composition
Novel method previously reserved for study of hard materials
Looking at variations in membrane composition is of interest to structural
biologists. However, investigating lateral variations caused by interactions of
membrane components — which are believed to play a crucial role in a range
of cellular events — has proved challenging, as achieving the necessary length
scales of tens to hundreds of nanometers can be difficult with the technologies
currently used for such probes.
These lengths of interest are well below the diffraction
limit of light microscopy. Researchers often use fluorescence microscopy to explore
labeled components, but the labeling process can alter significantly the physical
properties of molecules (especially those of small molecules, such as lipids, that
interact weakly). And although atomic force microscopy can provide much higher resolution,
it does not shed any light on the chemical composition of the membranes.
The lateral variations posed a problem
to a group at Stanford University in California: How do you image and acquire compositional
information about these variations with a higher spatial resolution than is available
with conventional light microscopy, with high sensitivity and in a nonperturbative
way? This led the researchers to various kinds of imaging mass spectrometers and,
specifically, to secondary ion mass spectrometry, principal investigator Steven
G. Boxer said. The scientists reported their use of the latter technique to analyze
phase separation of lipid membranes in the Sept. 29 issue of Science.
Secondary ion mass spectrometry could help shed light on a variety of questions; e.g., whether
reorganization of membrane components associated with one another occurs collectively.
In addition,researchers at Stanford University hope to improve the sensitivity of
the technique to investigate membrane-anchored proteins and, perhaps, to detect
lateral variations of individual labeled proteins in real cell membranes trapped
on substrate surfaces.
With secondary ion mass spectrometry,
a focused primary ion beam is rastered across a sample. Sputtering produces secondary
ions. These are collected, and the respective charge-to-mass ratios are analyzed.
Users can acquire secondary ion images characteristic of individual membrane components
by incorporating distinctive stable isotopes into them. From these they can derive
a component-specific compositional map of the sample.
The technique was developed —
and is typically used — for the study of hard materials such as minerals (as
opposed to soft materials such as cellular membranes). Generally, it uses a number
of types of beams — often pulsed ones, as are used for time-of-flight secondary
ion mass spectrometry. For this study, the researchers used a NanoSims 50 secondary
ion mass spectrometry system made by Cameca Instruments of Courbevoie, France.
“The NanoSims is quite special,”
Boxer said, “as it uses a continuous ion beam made of cesium ions that can
be very tightly focused.” This provides very high spatial resolution and enables
rather complete destruction of the molecules on the surface of the sample because
of the energy and nature of the beam. Conversion to secondary ions with high yield
enables high sensitivity.
Lawrence Livermore National Laboratory
in Livermore, Calif., maintains the secondary ion mass spectrometry system, which
is used by a variety of labs for study of dust collected from comets, among other
applications. Fortunately, Boxer said, some scientists who otherwise focus on hard
materials were willing to take a gamble with respect to collaborations on investigations
of soft materials and biology, setting the stage for broad application of the existing
technology to a whole new class of problems.
They used the spectrometry system to
measure the chemical composition of a phase-separated lipid bilayer, relying on
isotopic labels to identify each molecular species. The bilayer contained gel and
fluid phases, made up primarily of 13C18-DSPC (a phospholipid with long stiff chains)
and 15N-DLPC (a phospholipid with shorter chains), respectively. They used 13C1H—
and 12C15N— secondary ion signals to determine the distribution of each of these,
enabling construction of a component-specific compositional map of the bilayer,
which showed that it was not homogeneous.
To validate their findings, they characterized
the lipid bilayer with atomic force microscopy prior to analysis with secondary
ion mass spectrometry. Comparison of the images revealed that the geometries were
almost the same. In fact, the secondary ion images included phase-separated domains
with complex structures and domains, on the order of approximately 100 nm in diameter
according to the atomic force microscopy images. Beyond this, the study showed
that the scientists could quantify the lipid composition within small areas of the
bilayer — helping to determine whether the variations in 15N-DLPC distribution,
for example, were statistically significant.
Having demonstrated the feasibility
of measuring lateral variations in membrane composition with secondary ion mass
spectrometry, the researchers plan to return to the question that led them to the
technique: whether membrane components that are associated with one another —
in lipid rafts, for example — reorganize collectively.
“We’re sort of looking
up the ladder of complexity to see how far we can push the sensitivity issue,”
Boxer said. They plan to look at membrane-anchored proteins next. “But, conceivably,
we could detect down to the single protein level with appropriate labeling and move
on to real cell membranes and interfaces trapped on our surfaces,” he added.
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