Biology Reference
In-Depth Information
and adequate sample are available for addi-
tional analysis, then direct ion fragmentation
results can be easily obtained to aid in structure
elucidation. If not, ion fragments can be identi-
TABLE 4
Metabolic Pathway Websites
Name
Location
Reactome
fied through extracted ion monitoring methods
using existing spectra; for example, determine
the ions in the spectra that trace exactly together
and determine if logical losses can be assigned.
If logical losses can be assigned, then parts of
the unknown metabolite can be identi
KEGG
ExPASy
BioCyc
SGD
ed.
Based on LC retention time, mass fragmentation
of standards, and known structure fragmenta-
tion patterns, intelligent deductions can be
made regarding the structure of the unknown
metabolite. Additional re
Nutrition Metabolomics
Database
PMN
nements might be
necessary to further narrow the number of
possibilities. In many cases, high resolution LC
and GC can separate molecular isomers that
MS cannot distinguish. For additional aid in
metabolite
chemical shift ranges for common NMR active
nuclei are:
1
H ~12 ppm,
13
C ~220 ppm,
15
N
400 ppm, and
31
P
>
200 ppm. Different func-
tional groups have very characteristic chemical
shifts, and this information is extremely useful
for structure determination. For example, the
proton chemical shifts of saturated alkanes fall
in the ~0 to 2 ppm range and aromatic protons
are in the ~6 to 8.5 ppm range. Tabulations of
empirical chemical shifts for various functional
groups as well as proton
e
proton (
n
J
H,H
) and pro-
ton
e
carbon (
n
J
C,H
) scalar coupling values, where
n is the number of bonds, can be found in texts
such as Pretsch et al.
19
and Silverstein et al.
20
and the chemistry department of the University
of Wisconsin
e
Madison
>
cation, the reader may
consult the following references: for mass spec-
trum interpretation in general,
8,9
identi
lipids,
10,11
nucleic acids,
12,13
saccharides,
14
fatty acids,
bile acids, and steroids.
15
An identi
ed metabo-
lite, when placed into a metabolic pathway,
may lead to the identi
cation of additional
unknown metabolites belonging to the same
pathway. The reader is referred to the following
metabolite identi
cation and pathway sources:
energy metabolism,
16
choline metabolism,
17
and amino acid metabolism.
18
Anumberof
useful metabolic pathway websites are listed
in
Table 4
.
s website (
http://www.
Complementary information on functional
groups from IR and UV data is also often incor-
porated into the data analysis.
Complete structure determination by NMR is
based on chemical shifts, scalar couplings,
molecular connectivity, and spatial information
obtainable from a suite of 1D and 2D experi-
ments. The process starts with the systemic anal-
ysis of the 1D
1
H and
13
C (if available) spectra to
determine the number of hydrogens and carbons
present, chemical shifts, peak multiplicity, and
scalar coupling values. The number of hydrogen
'
NMR Metabolite Identi
cation
Metabolite identi
cation, whether it is struc-
ture con
rmation or de novo, is the core strength
of NMR spectroscopy. The NMR frequency of
a nucleus is dependent on the nuclear isotope,
the local electronic environment, and the
eld
strength of the NMRmagnet. NMR peak frequen-
cies are usually reported in parts per million
(ppm) based on a chemical shift scale that is inde-
pendent of magnetic
field strength. Typical
Search WWH ::
Custom Search