Biology Reference
In-Depth Information
trials. Finally, a clinical assay is developed for
a biomarker and subjected to approval by regu-
latory health agencies.
In vitro
diagnostic assays
for more than 200 unique proteins are currently
approved by the FDA,
12
and the majority of
them are based on ELISA. There is no a single
mass spectrometry
e
based protein assay used
in the clinics yet,
13
but a lot of efforts are
currently aimed toward the introduction of
such assays into clinical practice.
14
e
16
In the quest for noninvasive diagnostic
protein markers, urine is an attractive biological
fluid, given that it can be collected noninvasively
and in large quantities. Although urine proteo-
mics has been widely explored for identi
cation
of biomarkers related to renal or urogenital
disorders, other health conditions such as cancer
and in
ammation in distant organs may also
result in changes of the urine proteome.
21,22
Even though urine contains fewer proteins
compared to plasma, the urine proteome is still
complex, with more than 1,500 proteins identi-
PROTEOMIC SAMPLES
fied in healthy individuals.
23
Another challenge
of urine is the need for normalization and stan-
dardization of protein levels across different
samples. Protein concentrations in urine depend
on the glomerular
The choice of the proteomic sample suitable for
biomarker discovery study depends on a speci
c
clinical question addressed, sample availability,
and limitations of a biological model (
Figure 2
).
An array of proteomic samples can be used, but
blood plasma or serum are the most relevant bio-
logical
filtration rate and thus should
be normalized against reference molecules such
as creatinine.
24
The Human Kidney and Urine
Proteome
(HKUPP),
25
Project
a Human
fluids for biomarkers intended for
screening, diagnostic, or surveillance applica-
tions. Blood is the most abundant body
Proteome Organization
(HUPO)
e
sponsored
scienti
c initiative, provides guidelines for stan-
dardized collection and storage of urine samples
along with protocols for urine sample prepara-
tion and aims to construct a reference database
of normal human urine.
Due to the challenges of biomarker discovery
in blood and urine, the potential of other biolog-
ical specimens is being widely explored. Primary
sites of disease such as tissues and proximal
uid
and is easily collected by venipuncture, a proce-
dure with minimal invasiveness. Given that all
organs are perfused by it, blood re
ects the phys-
iologic state of the body at any time.
17
However,
the proteomic analysis of blood plasma or serum
is very challenging due to the wide dynamic
range of protein concentrations exceeding ten
orders of magnitude and the presence of lipids
and salts. The range of protein concentrations in
blood exceeds the dynamic range of mass spec-
trometry analyses by
fluids are attractive alternatives for biomarker
candidate
identi
cation
and
veri
cation.
Commonly used proximal
fluids include ascites,
five or six orders of magni-
tude.
18,19
Low-abundant proteins present in the
blood are usually masked by high-abundance
proteins, 22 of which constitute 99% of the total
protein mass.
18
In addition, physiological
concentrations of salts and lipids interfere
with mass spectrometry
e
based analysis.
20
Depletion of high-abundance proteins and
extensive fractionation may improve detection
of low-abundance proteins but at the cost of
decreased throughput and reproducibility of
analysis.
cerebrospinal
fluid, seminal plasma, expressed
prostatic secretion, nipple aspirate
fluid, saliva,
tears, pancreatic juice, and others. Proximal
fluids,
such as ascites
fluid in pancreatic and ovarian
cancers
26
often enclose the site of the disease
and accumulate disease-speci
c proteins result-
ing in their higher concentration relative to blood.
For example, median levels of CA-125, an
ovarian cancer biomarker used in the clinics,
were found as 696 and 18,563 U/mL in serum
and ascites
fluid, respectively.
27
Proximal disease
fluids, however, are usually collected through the
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