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Figure 1. Statistical total correlation spectroscopy (STOCSY): (A) one-dimensional (1D) and (B) two-dimensional (2D) STOCSY correlation/
covariance plots of a urinary NMR data set. Here, lineshapes are calculated by covariance ([,]) while colors are set by correlation ([1,1]).
One-dimensional STOCSY plots (A) are traces of the two-dimensional STOCY (B, black box) at a given chemical shift taken from the driver peak.
For the maxima of the doublet of 3-hydroxybutryic acid at 1.2 ppm (driver peak), high positive correlation coecients are both sensitive and
specic indicators of structural connectivity, and pathway connectivities show mostly negative correlations. For other metabolite signals seen in the
2D STOCSY, high positive correlations such as those between lactate (1.33 ppm), alanine (1.48 ppm), and glucose (3.54 ppm) indicate
coordinated excretion rather than structural relationships.
5298
Analytical Chemistry
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rmn =
i = 1 (xim xm )(xin xn )
(j 1)xmxn
(1)
R=
1
X1t X 2
j1
(2)
R=
1
X1t Y
j1
(3)
Analytical Chemistry
Perspective
Table 1
statistical spectroscopic tool
analysis platform
purpose
ref(s)
14, 33
46
37
47
44, 45
35
42
43
41
H/1H NMR
H/1H NMR
1
1
H NMR/LCMS
H/19F, 1H/31P NMR
1
H/1H NMR
1
H/1H
H/1H
1
H/1H
1
H/1H
1
NMR
NMR
NMR
NMR
H/1H NMR
H/1H NMR
1
H/1H NMR
1
36
76
38
40
39
Many of these discovered associations make immediate biological sense. Many cis associations between polymorphisms
in metabolic enzymes and adjacent metabolites (urine glycerate
and glycerate kinase54) and transporters and transporter
substrates (serum urate and SLC2A9 urate transporter58) are
immediately comprehensible. Other pathway associations can
be more dicult to explain at rst but are no less informative.
These associations are likely to annotate unknown gene
functions and help explain human metabolic networks in both
physiological and pathological states. The nearly simultaneous
discovery of a statistical association between the NT5E gene,
encoding a vascular epithelial enzyme, and serum inosine concentration58 and the identication of a human vascular calcication phenotype cause by rare mutations in the NT5E gene
and resulting disruption of adenosine metabolism61 indicates the
potential power of this approach.
In addition to mammalian genomic determinants of metabolism, the global systems biology approach enables the
identication of gut microbiota impacts on host metabolism.
Sensitivity to intestinal microbes is especially important for
organism-level networks as our microbiota includes over a
thousand species of symbionts with a total genetic contribution
of probably more than 100 times the 20 000 predicted human
genes. This second genome coordinates extensively with host
metabolic and physiological systems, especially the liver and
immune system.62 Associations of microbe populations with
metabolites have been enabled by semiquantitative characterizations of bacterial species using techniques such as uorescent
in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) and have been used to identify associations
between bacterial populations, related metabolites, and obesity.63
While extensions of statistical spectroscopy to nonmetabolite
data sets is a promising area of research, weaker associations
between levels of biological organization (e.g., gene regulation
and metabolite abundance) require increased statistical power
to detect. Population-level studies are increasingly necessary to
identify signicant associations in top-down systems biology.
The recently established MRC-NIHR National Phenome
Centre in the U.K. will play an important role in developing
the scale necessary to do this kind of science.
Analytical Chemistry
Perspective
Figure 3. Strategy for unknown compound identication using statistical spectroscopy. Signals of interest are identied from an experimental data set
(a). Statistical spectroscopy tools, such as STOCSY and SHY, are used to identify correlations between the signal of interest and additional signals in
the same or other spectral data sets collected from the same set of samples (b). Candidate compounds are identied using database search tools to
match the set of correlated spectral features with structures (c). A candidate compound is conrmed by spiking a solution of the candidate into the
original mixture (d).
Analytical Chemistry
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AUTHOR INFORMATION
Corresponding Author
*E-mail: j.nicholson@imperial.ac.uk.
Notes
ACKNOWLEDGMENTS
S.L.R. acknowledges support from the NSF Graduate Research
Fellowship Program and from the Marshall Aid Commemoration Commission.
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