Biology Reference
In-Depth Information
Unselective
Fast and minimal
handling
Reproducible
Metabolism
quenching
Number of
metabolite
features
Efficiency of
removal of
interferences such
as proteins
Good solubilization
of wide range of
metabolites
Compatibility with
analysis method
Sensitivity
Recovery studies
across different
metabolite classes
Minimize
metabolite
degradation during
preparation
Minimize
introduction of
exogenous
interferences
Adsorptive losses
Automation and
high throughput
Effect of short and
long term storage
Effect of handling
on distribution of
observed m/z
values for a given
metabolite
RSD distribution of
metabolite
features
In-depth
evaluation of
ionization
suppression
Control of critical
analytical and
preanalytical
factors
Identification of
metabolites with
high variability and
investigation of
root causes of
observed
variability
Comparison of
in
vivo
versus
ex vivo
metabolite profiles
Evaluation of
residual enzymatic
activity by
monitoring
substrates and
products in known
active pathways
Evaluation of
glutathione,
adenylate and/or
other relevant
ratios
Spiking and
recovery studies
FIGURE 1
Summary of characteristics of an ideal sample preparation method for untargeted metabolomics, including an
overview of parameters to consider during method development and evaluation. Parameters shown using black text have
been studied in literature, but limited or no information is available for parameters shown in blue text. (
Figure reprinted from
ref.
10
, with permission from Springer Science
þ
Business Media. Copyright 2012.
)
a comprehensive list of parameters that should
be evaluated to see how well a proposed method
meets the requirements of untargeted metabolo-
mics and to better understand the effect that the
chosen sample preparation method may have on
accurate biological interpretation. Unfortu-
nately, many of these parameters have not yet
been fully evaluated in terms of untargeted
metabolomics, leaving numerous gaps in our
knowledge, so fully rational design of global
sample preparation methods is currently not
possible and is currently an active area of
research.
In contrast to targeted analysis of
apriori
selected analytes, the goal of global metabolo-
mic sample preparation is to maintain the orig-
inal metabolite composition of the sample as
much as possible while also reproducibly trans-
forming the sample into a format that is
compatible with subsequent NMR or MS anal-
ysis.
Figure 2
highlights the main parameters
to consider and/or incorporate into the design
of sample preparation procedure for each of
the commonly employed analytical techniques
and sample types while attempting to preserve
metabolite composition of the original sample.
From a biomarker discovery perspective, one
of the most contradictory requirements is the
need for metabolism quenching, which is
routinely incorporated in cell, plant, and tissue
metabolomics but is generally omitted for
studies of biological
fluids such as blood. A
metabolism quenching step aims to stop meta-
bolic processes through use of low temperatures
(cold solvent addition, freezing in liquid
nitrogen), addition of acid, or fast heating.
14
e
18
However, metabolic processes can be very fast
with timescales of less than 1 s (for example,
ATP, glucose-6-phosphate, adenosine), so it
may be very dif
cult to implement an adequate
quenching step within the appropriate time
scale.
14
The addition of a quenching step can
itself cause inadvertent degradation or loss of
some metabolites, so it needs to be carefully
designed. For instance, acidic treatments can
cause severe degradation of some metabolites,
poor overall metabolite coverage, and poor
compatibility with MS methods.
5
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