Biology Reference
In-Depth Information
between two comparative samples. In the case of
samples obtained from healthy and disease-
affected individuals, the aim is to identify
disease-related differences in one or more of
the numerous endogenous metabolites found
in clinical samples such as bio
proteins. The cleavage of these polymers into
smaller units by enzymes makes possible their
analysis and identi
cation by MS; because these
molecules (polymers) are made up of known
monomers of speci
c molecular weights, the
MS results can be searched against a data base
of known composition and molecular weights
for sequence order and molecular identi
uids and tissue.
The analysis of a biological sample results in
hundreds of metabolites that need to be identi-
cation.
However, the structures of most metabolites
(small molecules) are made up of not repeating
monomer units but a random combination of
C, H, N, O, S, and P. Anabolic and catabolic
metabolite structure classes include saccharides,
simple sugars, lipids, steroids, isoprenoids,
porphyrins, purines, pyrimidines, amino acids,
catacholamines, acids, amines, and many
synthetic and natural exogenous substances. A
high
fied, which is not an easy task. In this section,
we discuss MS metabolite identi
cation and
NMR metabolite identi
cation.
MS Metabolite Identi
cation
It is not easy to identify a metabolite in
a metabolome, especially low-level metabolites
that are at or slightly above the noise level or
are masked by other metabolites. Metabolite
identi
cations
(hydroxylation, methylation, epoxidation, esteri-
diversity
of
chemical modi
rmation in GC/MS and
LC/MS is based on retention time, molecular
weight, and fragmentation pattern or compar-
ison of the metabolites
cation and con
fication, glycosylation, oxidation, reduction, and
isomerization) is observed within a class of
metabolites that add to the structure complexity.
The dif
spectra and fragmenta-
tion pattern with those of pure compounds. For
nontargeted metabolomics, there is no single
screening approach that will provide complete
coverage due in part to poor ionization, low ana-
lyte concentration, or poor chromatographic
retention. The ab initio identi
'
culty and scale of the effort required
for the identi
cation of metabolites depend on
the scope of the study: targeted versus global.
A targeted metabolomic study concentrates on
a well-de
ned group of compounds
d
for
example, identifying the major metabolites of
a potential drug candidate or a known metabolic
pathway leading to toxicity or detoxi
cation of small
molecules (less than 1,500 AMU) relies on the
knowledge and expertise of the analyst and
available tools. The identi
cation of
a drug. If the search is for known metabolites,
the identi
cation of organic
molecules is complicated by the fact that
compounds may have the same molecular
weight but exist in different forms (isomers). In
this case, the isomers are resolved by chromatog-
raphy prior to identi
cation involves comparing the exper-
imental data, m/z, and retention time, with that
of pure standards. If the metabolites can be
predicted
d
for example, discovery of the metab-
olites of a new potential drug
d
then metabolite
identi
cation by MS.
cation of proteins, peptides, DNA,
and RNA by mass spectrometry is easier than
the identi
The identi
finding representative
standards and searching for the predicted
metabolites in the experimental data. However,
if the metabolites are not known or cannot be
predicted, as in the discovery of biomarkers
for a particular disease, the metabolite identi
cation involves
cation of low molecular weight
compounds because of the linear and repetitive
nature of their building blocks. DNA is com-
posed of 4 nucleotides; proteins are composed
of 20 amino acids arranged in different linear
orders. It is the monomer order that imparts
the biological signi
ca-
tion is much more dif
cult and would
involve multiple chromatographic separations
using
cance to the DNA and
different
column
chemistries
and
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