Biology Reference
In-Depth Information
homogeneous and reproducible coatings to be
achieved. Spray coatings can be used for
imaging any class of molecules at relatively
high spatial resolution; however, care must be
taken with any spraying technique to ensure
that the surface is not made too wet, which can
lead to delocalization of analytes.
Very high spatial resolution can be achieved
through the use of sublimation to deposit matrix
onto the tissue section. Powdered matrix is
heated under vacuum and is redeposited onto
a cooler sample target that is suspended above
the matrix. Crystals formed are typically less
than 1
t of robotic spotters is the
ability to deposit matrix or other reagents at very
speci
An additional bene
c locations within a tissue section, allow-
ing for an approach known as histology directed
pro
ling.
25
In this type of experiment, two
sections of a tissue specimen are collected, one
on a MALDI target and a serial section on a stan-
dard microscope slide for histological staining.
After a photomicrograph has been taken of the
stained section, the image is annotated by
a pathologist or biologist to indicate areas of
interest for a given sample. The annotated image
is then merged with an image of the unstained
serial section using photo processing software
and the coordinates of the annotations are deter-
mined. These coordinates are transferred to the
robotic spotter and matrix is applied only to
those speci
m and the coating is very homogeneous.
When conditions such as temperature, pressure,
and time are carefully controlled, sublimated
coatings are highly reproducible and are appli-
cable for the analysis of most classes of lipids,
as no solvent is needed for extraction. If larger
molecules such as peptides and proteins are to
be analyzed, the use of a rehydration/recrys-
tallization step is employed to help cocrystalize
these molecules with the matrix.
24
Matrix and other reagents can also be applied
to the tissue through the use of robotic spotters
such as acoustic spotters (Labcyte Portrait)
19
and chemical inkjet printers.
9
Although these
spotters cannot achieve the spatial resolution of
the uniformcoating technologies describedprevi-
ously (they are typically limited to ~150
e
200
m
c locations. Typically 10 to 20 areas
are targeted per cell type per tissue, with 10 or
more samples analyzed in a single experiment,
greatly reducing the volume of data collected,
increasing the throughput, and enabling more
facile statistical analysis. Histology directed
pro
ling can be thought of as a type of low-
resolution imaging through correlation with
a microscopy image. In this way, the spatial
localization of analytes is preserved while gener-
ating high-quality data.
m
spot size), they tend to have the highest reproduc-
ibility of any of the coatings due to the tight
control of the exact volume of reagent applied
to a speci
m
PROTEIN ANALYSIS
Imaging mass spectrometry has been used for
the analysis of proteins in cancer tissue includ-
ing breast,
26
e
30
prostate,
10,31,32
kidney,
33
skin,
34
colon,
35
lung,
36
e
40
ovarian,
41
and gastric
42,43
as
well as other diseases such as in
c location on a surface. Spotting gener-
ally leads to the most robust analyte signal due to
the ability to thoroughly wet small areas of
a tissue section for increased extraction without
the risk of delocalization that occurs with spray
or rehydration techniques. Because of the small
reservoir volume (hundreds of microliters) of
commercial spotters, they may also be employed
for the application of relatively costly reagents to
the tissue section including enzymes for proteo-
lytic digestion and internal standards for drug
analysis and quantitation.
ammatory
bowel diseases,
44
S. aureus
infection,
45
and
sarcoidosis.
46
The aim of most of these investiga-
tions is the identi
cation of proteins and protein
signatures for improved diagnosis, prognosis,
prediction of response to treatment, as well as
determination of potential drug targets.
IMS has been used in the classi
cation of
papillary bladder tumors into high grade (HG)
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