Biomedical Engineering Reference
In-Depth Information
The major strengths of MALDI imaging are in the quantification of intact protein species
directly from intact tissue sections where the spatial/topological distribution is retained.
As with the DIGE technology described in Section 13.2.1, large datasets can be easily
normalized and analyzed using multivariate statistical analyses that enable the visualization
of variation between samples as well as identify prognostic and diagnostic indicators of
phenotype. This technique generally consumes small sample amounts relative to the other
technologies (e.g. see Ref. [65]), although this will vary depending on the experiment.
The major limitation of MALDI imaging is that only those proteins that are soluble under
matrix application conditions that retain the
in vivo
spatial protein distribution are analyzed.
In addition, the identification of proteins occurs in a separate experiment, usually after
biochemical enrichment/fractionation, although methods to identify proteins directly from
tissue specimens are progressing [66].
13.3 Troubleshooting
No single proteomics technology platform is capable of accomplishing a true global anal-
ysis of the entire proteome. Each of these major technology platforms has strengths and
limitations, and in many cases these complement each other across platforms. Ultimately,
all analytical platforms have fundamental limits in total protein amount before resolution
is affected, and this is constrained in some sample types by the presence of a few proteins
comprising the bulk of the sample (this is most problematic in serum/plasma studies [67]).
The numbers provided in the following comparisons are approximate and reflect typical
ranges, but of course will be dependent on the nature/quality of the samples being analyzed.
13.3.1 Number of resolved features and modifications
All three of the major technology platforms discussed here have the ability to provide for
resolution on hundreds to thousands of features. The majority of intact signals tractable by
the MALDI-IMS technology typically fall below the 30 000 MW range, and this technology
retains the spatial distribution within intact tissues. The intact features that can be resolved
in sample homogenates by 2D gels for the DIGE technology typically fall within the MW
range of circa 10-200 kDa, and the isoelectric focusing range of pH 3-11. Both DIGE/MS
and MALDI-IMS analyze and quantify intact protein species, and both are amenable to the
analysis of independent (biological) replicates across multiple experimental conditions to
enable facile multivariate statistical analysis. In some cases (e.g. phosphorylated proteins),
modified isoforms can be directly visualized by the 2D gel pattern (a charge-train of proteins
in the first dimension) and easily verified by protein excision, in-gel digestion and MS
(even without MS information on the modified peptide(s) per se). Such isoforms would
be apparent in the MALDI-IMS experiment as a series of
m
/
z
peaks (e.g. offset by 80 Da
for phosphorylation). Unless these modified peptides were readily detected (or specifically
targeted) in an LC/MS/MS experiment, they may be difficult to capture/analyze in a complex
mixture (especially in a global-scale discovery study) from a bottom-up shotgun analysis.
Proteins that are 'difficult' to resolve by intact-protein methods are often readily identified
using the LC/MS/MS shotgun approach because sufficient surrogate peptides can be obtained
and mass analyzed (assuming that the proteins remain soluble during the various isolation,
extraction and digestion steps). The shotgun approach also provides for greater sensitivity
and dynamic range, especially via MudPIT analysis. However, when using peptide-based
