University of Aveiro

Abstract
Malaria affects an estimated 216 million people worldwide and is responsible for the
death of approximately 600,000 individuals annually, most of them children from
Africa.
The
most
current
standard
M., Lopes, D.
method
of diagnosys is through a microscopy visualization of the parasite in a blood sample. In
this article we propose a optimized protocol to the utilization of proteomics in the
identification of possible malaria biomarkers present in the saliva of the patients
through the depletion of highly abundant proteins present in saliva such as amylase
and lectins, in order to improve the dynamic range of the protein identification.

Salivary proteomics in the identification of parasite biomarkers

in patients with uncomplicated plasmodium falciparum malaria
Sousa,

Keywords: malaria, salivary proteomics, iTRAQ

Introduction
Malaria is an important public health
issue causing over 216 million cases
worldwide and nearly 655,000 deaths
annually [1].
Charles Laveran in 1880 first visualized
the protozoan parasite that causes
malaria in a smear of blood under a
microscope, and from the early decades
of the 20th century to date, microscopy
has been the standard method to
detect malaria [2].
In laboratories throughout the world,
routine diagnosis is carried out by thick
blood film microscopy, which is up to 10
times more sensitive than thin blood
(smear) film microscopy [3], and its
used for detecting malaria parasitemia
and estimating its level [4]. The current
methods of detection are invasive and
associated with poor compliance when
repeated sampling is required, for
instance PCR. So new approaches need
to arise to fill these gaps.

Saliva started for being less studied

than other body fluids, but in the last
years it has being receiving an
increasing amount of attention [5].

Salivary secretions are a highly complex

mixture of proteins such as hormones,
enzymes, lipids, carbohydrates and ions
that contribute to the many roles and
functions of saliva [1]. The composition
of saliva results from the contribution of
the salivary glands, oral tissues and oral
micro-organisms [6]. One decade after
the introduction of proteomics and the
following
advances
in
mass
spectrometry, a tremendous progress in
disclosing
the
complete
salivary
proteome was noticed, which have lead
in more than 3000 different proteins
already been identified [7].
It was the discovered that saliva
contains molecular profiles that reflect
systemic diseases, which opened the
doors to a new non-invasive emerging
diagnostic methodology [8].

Saliva has the advantage of being a

readily accessible, non-invasive body
fluid [1]. So if assays that use these
non-invasive obtained samples, like the
case of salivary proteomics, prove to
have high diagnostic accuracy, malaria
could be diagnosed more accurately
because samples could be obtained
more
frequently
with
minimal
inconvenience to patients [9].
The significant overlap in protein
content between saliva and plasma
suggests that saliva could be a
potentially attractive fluid for disease
biomarker discovery as well as a
diagnostic alternative to blood tests
[10,11],
such
as
in
human
immunodeficiency
virus
(HIV),
or
systematic diseases that affect the
function of salivary glands, e.g.,
Sjgrens syndrome, alcoholic cirrhosis,
cystic
fibrosis,
diabetes
mellitus,
diseases
of
the
adrenal
cortex,
cardiovascular diseases, and dental
caries [12-14]. A thorough knowledge of
whole saliva is a determinant pre-factor
for an accurate and precise use and
should be explored [15] for a viable
correlation with the parasites loads
detected in blood [9].
The complexity and the high dynamic
range of salivary proteins are the main
adversities found in identification of
potential biomarkers in saliva using
mass spectrometry as evidenced by
highly-abundant
proteins
such
as
amylase, which constitutes about 60%
of the protein composition of saliva
[6,16,17]. Therefore, the depletion of
highly abundant proteins prior to mass
spectrometry analysis is a critical step
to maximize the detection of less
abundant proteins that may be
differentially expressed in response to
malaria infection [1]. Lectins are
carbohydrate-binding
proteins
that
reversibly bind to specific mono- and
oligosaccharides
[18,19].
The

reversibility
of
the
lectin-sugar
interactions makes them appropriate for
enrichment/depletion strategies [18]. In
this context, the differential binding
affinities of lectins makes them an
effective
tool
for
glycoproteomic
research. For example, concanavalin A
(ConA) binds with high mannose type Nglycans,
whereas
jacalin
(Jac)
selectively
binds
immunoglobulins
[20,21].
This
protocol
addresses
the
methodological process to be used in
the use of salivary proteomics in the
identifications of possible malaria
biomarkers through the depletion of
highly abundant proteins from saliva
such as amylase and lectins.

Methods

Notes (additional files)

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