Biomedical Engineering Reference
In-Depth Information
•
Can be coupled with a second chromatographic separation (e.g. strong cation exchange)
for MudPIT for very complex mixtures with high sensitivity and dynamic range.
•
Isotope-labeled protein/peptide tags (e.g. ICAT, iTRAQ) or label-free spectral counting
techniques for quantitative analysis.
As LC/MS-based strategies are performed on peptides derived from intact proteins, this
technology generally delivers the most sensitive survey of proteins present in a sample owing
to the fact that peptides are generally homogeneous in their physicochemical properties
and can be resolved in time using HPLC, and often only a handful of peptides may be
necessary to identify a protein unambiguously. Furthermore, the additional resolving power
of multiple HPLC separations during MudPIT experiments affords excellent sensitivity and
dynamic range [2, 3].
The limitations of LC/MS-based technologies are in the ability to easily distinguish
between post-translationally modified isoforms and/or proteolytic cleavage products, as well
as lower statistical power for global/discovery quantitative studies. This is mainly due to
the fact that the analysis is performed on the peptide products of the starting mixture
en
masse
without any measurement of the intact proteins, such that isoforms remain unresolved
and proteolytic products are masked by the intact species if both are present. The lower
statistical power stems mainly from the fact that the mass spectrometer is sampling ions
for tandem MS based on relative signal intensities that can vary from run to run [40, 41],
requiring many technical replicates for each independent sample to attain acceptable levels
of statistical power.
13.2.3 MALDI imaging and profiling
A main feature of MALDI imaging mass spectrometry (IMS) is that it is able to map the
spatial distribution of proteins within intact tissue specimens and produce molecular images
of specific protein species. This is in contrast to the 2D-gel based and LC/MS/MS-based
technologies that are performed on protein homogenates, where this spatial information is
not retained. With MALDI-IMS, samples such as thin sections cut from fresh-frozen tissue
biopsies [42-44], cells procured via laser capture microdissection [45] and fluids [46] can
be analyzed directly in the mass spectrometer (Figure 13.3). As is the case for the 2D-gel
technologies, MALDI-IMS also quantifies expression at the level of intact proteins (where
modified and proteolytic forms are resolved), and is directly amenable to complex experi-
mental designs including independent replicates from multiple experimental conditions, as
well as multivariate statistical analysis. This process can resolve and analyze intact molecu-
lar signals from a single MS acquisition within a tissue section (e.g. as small as 25-50
μ
m
laser diameter in the MALDI source) [47].
In most cases, cryostatically derived tissue sections are mounted onto a MALDI target
plate, prepared for MS on-target and introduced directly into the ion source (reviewed in Refs
[48, 49]). This type of experiment can be performed in low-resolution 'profiling' mode where
the tissue of interest is interrogated in multiple discrete locations (often directed by histology
[50]), or in high-resolution 'imaging' mode whereby MALDI-TOF spectra are acquired at
every position along a rastered
X
-
Y
array across the tissue. In such an imaging experiment,
individual mass/charge (
m
/
z
) values (each representing a discrete intact molecular species)
can be tracked throughout a tissue section where relative ion signal is proportional to protein
expression. This generates an ion density map for each resolved
m
/
z
species in the analysis.
The resulting molecular profiles and high-resolution images can be compared within and
