Biology Reference
In-Depth Information
molecules within a tissue section but requires
that the speci
that can be extracted from the tissue surface.
Other lesser-used approaches include laser abla-
tion electrospray ionization,
11
matrix assisted
laser desorption electrospray ionization,
12
and
jet desorption electrospray ionization.
13
These
latter techniques will not be discussed in detail
in this chapter and the reader is referred to recent
reviews
14,15
for more information.
In the present chapter, we will focus on the
use and application of matrix-assisted laser
desorption/ionization imaging mass spectrom-
etry (MALDI IMS) as the most broadly appli-
cable approach for the analysis of a wide
variety of biomolecules. MALDI IMS has been
used for the analysis of proteins, peptides, lipids,
drugs, and metabolites from tissue specimens
ranging in size from cell clusters
16
to whole
animal sections.
17
In the MALDI approach,
a matrix (typically a small organic molecule) is
applied to the surface of a tissue section in
a solvent that aids in the extraction of analytes.
The speci
c analyte of interest be known in
advance and that an antibody-based reagent
against it must exist. Additionally, only a few
molecules can be probed from a single tissue
section. Conversely, IMS is carried out without
the need for homogenization, allowing for the
analysis of hundreds to thousands of biomole-
cules in their native locations within a single
tissue specimen. This technology is excellent for
discovery because no target-speci
c reagents
such as antibodies are required.
Several surface ionization techniques can be
used to generate data for imaging applications.
One of the early approaches reported is
secondary ion mass spectrometry (SIMS), in
which a surface is bombarded with a primary
ion beam, leading to desorption of molecules
and generation of secondary ions.
4
e
6
Although
the SIMS approach allows for very high spatial
resolution imaging (
m), the high-energy
primary ion beam is destructive to surface
biomolecules generated, effectively limiting the
practical mass range of analysis to less than
1,000 Da. Desorption electrospray ionization
(DESI) uses a capillary spray of solvent onto
the surface of the tissue to desorb ions from
a tissue surface that can then be introduced
into a mass spectrometer through a second inlet
capillary.
7
e
9
Advantages of the DESI technique
are that it can be used at ambient pressure and
requires minimal sample preparation. However,
it is most effective in analyzing small molecules
such as drugs and lipids and spatial resolution
is typically 100
1
m
<
c matrix and solvent used can be
optimized for different classes of molecules.
15
A raster of the tissue surface by the laser induces
the desorption and ionization of molecules from
the tissue surface (
Figure 1
). Commercial instru-
ments allow for achievable spatial resolutions of
approximately 20
cations
through use of specialized optics have allowed
for spatial resolutions of approximately 1
m
m, and custom modi
m.
18
As usual, the trade-off between spatial resolu-
tion and sensitivity ultimately determines
the actual
m
resolution employed in a given
experiment.
m
m or more. Liquid extraction
surface analysis (LESA) uses a small volume of
solvent applied to an area of interest on a tissue
section using a pipet, which is then drawn
back up into the tip.
10
The extracted molecules
are separated by rapid liquid chromatography
and detected via mass spectrometry, often by
selected reaction monitoring. The LESA tech-
nique is limited by low spatial resolution; tar-
geted areas are often on the order of 1 to 2 mm
but could be applicable to any type of molecule
MATRIX APPLICATION
A variety of techniques can be used for the
application of matrix to the tissue surface and
the method of choice depends on the imaging
task that is to be done. Wet extraction methods
such as spotting
19
and spraying
20
result in
a higher degree of analyte extraction, especially
for larger polar molecules such as proteins, but
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