Biomarker Discovery: Study Design and Execution - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 9

Sample collection, handling, and storage have
great impact on the sensitivity, selectivity, and
reproducibility of any given analysis. Detailed
information on clinical and pathological parame-
ters should be secured before samples are
collected. Specimens should be collected by
trained personnel. Blood samples should immedi-
ately be converted to serum or plasma and stored
in the freezer at e 80 C until time of analysis to
prevent any enzymatic activity that can result in
inconsistent protein degradation or metabolite
interconversion. Two studies have shown a signif-
icant effect of freeze/thaw cycles on the proteome
pro

Sample Preparation
Preparation of the sample for proteomic and
metabolomic analysis can introduce errors that
will affect the quality of the

final results. The
search for biomarkers in biological samples
involves different steps depending on the
sample type and whether the analysis is for
metabolites or proteins,

targeted or global

(pro ling). Extraction of metabolites from blood,

urine, or tissue for a global study is not an easy
task. It may require multiple extraction proce-
dures using different solvent systems. It is not
always possible to extract all the metabolites
from a sample with a single solvent because
metabolites have different chemical and physical
properties and are present in a wide dynamic
concentration range. For details, see Chapters 3
and 4 on sample preparation for proteomics
and metabolomics within this topic.
Preparation of a blood sample for proteomic
study is more complicated than urine, as urine
contains fewer proteins and cells. The high
abundant proteins must be depleted from blood
prior to HPLC/MS/MS analysis. Approxi-
mately 99% of the protein content of blood
(both serum and plasma) is made up of only
about 20 proteins. 25 Although depletion of these
proteins allows for the detection of low abun-
dant proteins, it also removes proteins that are
bound to these 20 proteins, resulting in the
loss of potentially important information. 26
Tissues are initially homogenized prior to
extraction of metabolites and proteins. Incom-
plete homogenization can lead to losses that
can affect the accuracy and precision of the
results. For detailed discussion, see Chapters 3
and 4 on sample preparation for metabolomics
and proteomics.

le of serum/plasma. 19,20 Also, factors
utilized in the preparation of serum, such as the
anticoagulant used, the clotting time allowed,
and the length of the time period before centrifu-
gation, had a signi cant effect on the serum pro-

teome. A few studies have been carried out
showing that sampling procedures (i.e., fasting,
time sample acquired frompatient) had the great-
est effects on proteome pro

ling, while handling
procedures and storage conditions had relatively
minor effects. 21 However, everyone agrees that
standardized protocols for sample, handling,
storage, and analysis are required, as the issue is
not about which procedure is better but rather
about using standardized procedures to obtain
comparable and reproducible results between
different laboratories. 22 e 24

Method of Sample Analysis
Selection of the method of sample preparation
and analysis plays an important role in deter-
mining the accuracy of the results, as discussed
later in this chapter.

ER RORS IN STUDY EXECUTI ON

Methods of Analysis
Choosing the optimal analysis method is crit-
ical in proteomic and metabolomic studies.
For example, analyzing the plasma proteome

Study execution deals with many experi-
mental parameters that should be carefully
considered for a successful experiment with
meaningful and reproducible results.