Biology Reference
In-Depth Information
Metabolite derivatization procedure for GC
analysis.
The identities of metabolites are generally based
on comparison of the experimental data to that
contained within databases, followed by compar-
ison to a pure compound of the same identity. The
comparison should involve more than one proce-
dure or parameter d for example, m/z ratio, reten-
tion time, sequence, NMR resonance.
￿
￿
Protein digestion conditions.
￿
Instrument quali
cation; ensure that all
instrumentation is operational and
performance quali
ed by a certi
ed
technician.
Operator training and quali
cation.
￿
Analytical procedures should be validated,
inter- and intraday variability, sample to
sample reproducibility and relative standard
deviation should be determined and their
acceptable levels speci
￿
BIOMARKER VALIDATION
Validation of a biomarker is a lengthy and costly
processthatinvolvesamu iphaseapproach.
Pepe et al. 2 suggested that the following
ed.
ve phases
Reproducibility: the results should be
reproducible in other labs, by different
personnel using different (but equivalent)
instrumentation.
￿
be carried out consecutively.
Phase 1: Preclinical exploratory studies
to identify potentially useful markers
This step involves the experimental identi
Blind testing and interpretation of results.
￿
Identities and purity of internal standards
should be con
￿
ca-
tion of a protein or metabolite as a discriminating
factor between diseased samples and controls.
Most published proteomic and metabolomic
studies stop at this phase creating a glut of
potential biomarkers that languish at this phase.
rmed.
Determine whether different techniques
measure the same compounds quantitatively.
￿
Statistical data analysis method should be
speci
￿
ed.
Phase 2: Clinical assay development for
clinical disease
The aim of this phase is to ensure that the
candidate biomarker (protein or metabolite)
discriminates between diseased and control
samples that were obtained in a noninvasive
manner. This phase also involves optimization
of the analytical method. It is important to deter-
mine the effect of sex, age, and ethnicity of
controls on the biomarker discovery results while
assessing the ability to differentiate between
characteristics such as cancer stage and grade.
BIOMARKER IDENTIFICATION
AND CONFIRMATION
Analytical and statistical data determined that
certain proteins or metabolites are found to be
differentially expressed in the biological
fluid or
tissue of the patient subjects compared to healthy
controls. At this point, a rigorous investigation of
the results should be undertaken to con
rm the
identity of the potential biomarker. The analytical
methods of choice for the identi
cation of proteins
or metabolites biomarkers include LC/MS, gel
electrophoresis/MS, and NMR. In the case of
LC/MS, the compounds are identi
ed by their
Phase 3: Retrospective longitudinal
repository studies
The primary aims of this phase is to evaluate,
as a function of time before clinical diagnosis, the
capacity of the biomarker to detect preclinical
mass/charge ( m/z )
ratio and retention time.
Proteins are identi
ed by MS/MS, owing to the
invaluable sequence information it provides.
Because of isobaric compounds, metabolites may
not be as easy to identify as peptides and proteins.
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