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Peptidomics of Urine and Other Biofluids for Cancer Diagnostics


Abstract and Figures

Background: Cancer is a leading cause of death worldwide. The low diagnostic sensitivity and specificity of most current cancer biomarkers make early cancer diagnosis a challenging task. The comprehensive study of peptides and small proteins in a living system, known as "peptidomics," represents an alternative technological approach to the discovery of potential biomarkers for the assessment of a wide variety of pathologies. This review examines the current status of peptidomics for several body fluids, with a focus on urine, for cancer diagnostics applications. Content: Several studies have used high-throughput technologies to characterize the peptide content of different body fluids. Because of its noninvasive collection and high stability, urine is a valuable source of candidate cancer biomarkers. A wide variety of preanalytical issues concerning patient selection and sample handling need to be considered, because not doing so can affect the quality of the results by introducing bias and artifacts. Optimization of both the analytical strategies and the processing of bioinformatics data is also essential to minimize the false-discovery rate. Summary: Peptidomics-based studies of urine and other body fluids have yielded a number of biomolecules and peptide panels with potential for diagnosing different types of cancer, especially of the ovary, prostate, and bladder. Large-scale studies are needed to validate these molecules as cancer biomarkers.
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Peptidomics of Urine and Other Biofluids for
Cancer Diagnostics
Josep Miquel Bauc¸a,
Eduardo Martı´nez-Morillo,
and Eleftherios P. Diamandis
BACKGROUND:Cancer is a leading cause of death world-
wide. The low diagnostic sensitivity and specificity of
most current cancer biomarkers make early cancer di-
agnosis a challenging task. The comprehensive study of
peptides and small proteins in a living system, known
as “peptidomics,” represents an alternative technolog-
ical approach to the discovery of potential biomarkers
for the assessment of a wide variety of pathologies. This
review examines the current status of peptidomics for
several body fluids, with a focus on urine, for cancer
diagnostics applications.
CONTENT:Several studies have used high-throughput
technologies to characterize the peptide content of dif-
ferent body fluids. Because of its noninvasive collection
and high stability, urine is a valuable source of candi-
date cancer biomarkers. A wide variety of preanalytical
issues concerning patient selection and sample han-
dling need to be considered, because not doing so can
affect the quality of the results by introducing bias and
artifacts. Optimization of both the analytical strategies
and the processing of bioinformatics data is also essen-
tial to minimize the false-discovery rate.
SUMMARY:Peptidomics-based studies of urine and
other body fluids have yielded a number of biomol-
ecules and peptide panels with potential for diagnosing
different types of cancer, especially of the ovary, pros-
tate, and bladder. Large-scale studies are needed to val-
idate these molecules as cancer biomarkers.
© 2013 American Association for Clinical Chemistry
Cancer is a major clinical problem worldwide. Ac-
counting for approximately 1 in every 4 deaths, cancer
represents the second leading cause of death in devel-
oped countries, after cardiovascular diseases. It is esti-
mated that more than 1.6 million new cancer cases are
diagnosed every year in the US (1 ). Highly heteroge-
neous and with only a few effective therapeutic strate-
gies, cancer represents a challenging medical condition
for both healthcare professionals and governments.
The possibility of detecting cancer at early stages,
before it spreads to anatomically distant tissues, has
long interested physicians and scientists, because early
diagnosis is a key factor for successful treatment out-
comes. For instance, the overall 5-year survival rate for
ovarian cancer is 40%, whereas the rate increases to
90% if it is detected in its early stages (2 ). Conse-
quently, Finding new tools, such as endogenous
biomolecules, that could help identify patients with
early-stage disease is highly desirable. As stated by the
WHO in 1968, the ideal biomarker for a disease should
be measurable via a simple, reliable, and affordable
method and have a high diagnostic sensitivity and
specificity. The biomarker should be present in higher-
than-normal concentrations during early disease
stages, and its concentration should reflect the extent
or severity of the disease. Defining the target popula-
tion for whom the test would be applied is also of major
Unfortunately, only a few cancer biomarkers have
entered routine use. Even fewer have been approved for
population screening or diagnosis (3 ). One of the most
frequently used cancer biomarkers is prostate-specific
antigen (PSA).
Despite its widespread measurement,
many issues relating to overdiagnosis and overtreat-
ment have arisen because serum PSA also increases in
benign prostatic hyperplasia and other nonmalignant
diseases (4 ). Similarly and despite being considered the
best biochemical marker for breast cancer, carbohy-
drate antigen 15.3 is also increased in other tumors,
such as pancreatic and colorectal cancers, as well as in a
number of benign pathologies. The lack of diagnostic
sensitivity for this antigen, the serum concentrations of
Servei d’Ana` lisis Clı´niques, Hospital Universitari Son Espases, Palma de Mal-
lorca, Spain;
Samuel Lunenfeld Research Institute, Joseph and Wolf Lebovic
Health Centre, Mount Sinai Hospital, Toronto, Ontario, Canada;
Department of
Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario,
Department of Clinical Biochemistry, Toronto General Hospital, Uni-
versity Health Network, Toronto, Ontario, Canada;
Department of Pathology
and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.
* Address correspondence to this author at: Department of Pathology and
Laboratory Medicine, Mount Sinai Hospital, Suite 6-201, Box 32, 60 Murray St.,
Toronto, Ontario, M5T 3L9 Canada. Fax 416-619-5521; e-mail ediamandis@
Received June 21, 2013; accepted October 22, 2013.
Previously published online at DOI: 10.1373/clinchem.2013.211714
Nonstandard abbreviations: PSA, prostate-specific antigen; CSF, cerebrospinal
fluid; LC-MS/MS, liquid chromatography–tandem mass spectrometry; CA125,
carbohydrate antigen 125.
Clinical Chemistry 60:7
000 – 000 (2014) Reviews
1 latest version is at
Papers in Press. Published November 8, 2013 as doi:10.1373/clinchem.2013.211714
Copyright (C) 2013 by The American Association for Clinical Chemistry
which barely increase in early-stage malignancy, is also
an important limitation (5 ). Clearly, there is an urgent
need to discover and validate new biomarkers with bet-
ter performance characteristics.
High-throughput technologies that generate mas-
sive quantities of data have become known as “omics,”
a suffix derived from “genomics,” the comprehensive
study of genes and other DNA sequences (i.e., the
genome)— hence transcriptomics, proteomics, metabo-
lomics, epigenomics, and peptidomics. Three steps are
essential in the process of developing a biomarker (3 ):
(a) the discovery of candidate molecules in defined pa-
tient groups, (b) validation of the biomarkers for their
capacity to assist in disease assessment, and (c) imple-
mentation in the clinical setting.
This review focuses on the current situation of
cancer biomarker discovery through peptidomics. The
emphasis is on urine, but this review also covers inves-
tigations of other body fluids. Technological aspects of
peptidomics and their applications to different types of
cancer are also reviewed.
Proteomics and Peptidomics
Since the dawn of the genomics and transcriptomics
era, numerous efforts have been directed toward dis-
covering biomarkers that could help in the diagnosis,
prognosis, or monitoring of different diseases. The
main limitation of nucleic acid– based approaches is
that recognition of an inherited predisposition to dis-
ease is usually not sufficient to identify the biological
processes and mechanisms by which they operate (6 ).
This limitation can be partially alleviated with pro-
teomics. A complete analysis of the protein content of a
cell, tissue, or organism comprises all of the layers of
information gathered from the genome and the tran-
scriptome, plus posttranslational modifications (e.g.,
phosphorylation, glycosylation). Proteomics is the
large-scale study of the full complement of proteins in a
living system—their structures, their physicochemical
properties, and their functions. Proteomic technolo-
gies have the potential to detect dynamic changes in the
production of proteins via these technologies’ integra-
tion of the proteome’s genetic and epigenetic features
(7 ). The proteome is hence much more complex than
the genome or the transcriptome, and it appears to re-
flect actual cellular processes more accurately than the
genome or the transcriptome. Proteins are the effectors
of biochemical actions (Fig. 1). From a pathophysio-
logical point of view, genetic analyses can predict the
risk of developing a disease, whereas proteomic ap-
proaches have a capacity both to show when the risks
become evident as a disease and to facilitate monitor-
ing of the therapeutic response. Both the concentra-
tions of proteins and their posttranslational modifica-
tions may be altered during disease progression (8 ).
The peptidome constitutes the low molecular
weight proteome. The term “peptidomics,” a term
coined in 1996, is the systematic and comprehensive
analysis of the small proteins and endogenous peptides
of biological samples at a defined time. Peptidomics
typically encompasses polypeptides 20 kDa, al-
though no clear limit has been established. It is inter-
esting that results obtained with the first proteomic
methodologies appeared to indicate that the peptide
content samples was too simple and easily cleared by
the kidney to carry useful information. That turned out
not to be the case, for research efforts with peptidomics
have already yielded positive results.
Most peptides in biological systems are not syn-
thesized as such, but rather are derived from precursor
proteins via proteolytic cleavage by endogenous pepti-
dases in a specific or nonspecific way (Fig. 2), e.g., the
activation of some zymogens of the coagulation cas-
cade or the maturation of insulin. Other peptides are
generated in situ and then traverse the endothelial vas-
culature, if they are sufficiently small to enter the blood
passively or the wall becomes permeable owing to dis-
ease conditions (9, 10 ).
Proteolytic processing has been theorized to be
necessary to facilitate metabolic variation so that indi-
Fig. 1. Application of “omics” technologies for dis-
covering novel biomarkers.
Peptidomics may encompass additional sources of informa-
tion owing to proteolytic processing of proteins by active
2Clinical Chemistry 60:7 (2014)
viduals and species may better adapt to exogenous
stimuli (11 ). Actually, peptides in body fluids are be-
lieved to be due to an imbalance between the activities
of proteases on the one hand and the actions of pro-
tease inhibitors on the other. In this way, endogenous
proteases are differentially regulated in the contexts of
many physiological and pathologic phenomena (12 ).
Therefore, it is reasonable to hypothesize that studying
protease activity and regulation can lead to improved
detection and a deeper understanding of the molecular
mechanisms of some diseases.
Peptides also play central roles in healthy physio-
logical processes (13 ). That is the case for many cyto-
kines, growth factors, and some neuropeptides for
which proteome mapping studies have revealed no
precursor protein (14 ), suggesting that they are synthe-
sized as such in the central nervous system and are not
breakdown products of precursor proteins. Even pep-
tides processed from large proteins usually show bio-
logical functions and activities different from their par-
ent molecules (7 ). Given that a high percentage of
proteins undergo proteolytic cleavage, identifying and
characterizing their breakdown products might be of
interest, because they could be even more informative
than the precursor protein (10 ). The study of differen-
tial protease activities could be an inviting field for the
application of peptidomics to medicine. For example,
neoplastic processes are involved in the transformation
and proliferation of certain cell types and thus alter the
concentrations and activities of specific proteins and
enzymes, such as proteases. Therefore, not only do pro-
teins in the system (proteomics) become altered, but
their metabolic products (peptides), which should be
regarded as an extension of the proteome, also change.
Peptidomics of Body Fluids
The peptidome constitutes a still mostly unexplored
source of biological information, and it might provide
useful biomarkers for disease assessment. Peptides in
body fluids are proxies for protein synthesis, process-
ing, and degradation. Worth mentioning is that some
peptide biomarkers have already entered the clinic, al-
though none of them were discovered with contempo-
rary peptidomic methods (15 ) (Table 1). The peptides
most widely known are the aminoterminal propeptide
of brain natriuretic peptide (which is measured in se-
rum for assessing heart failure) and C-peptide (for
monitoring endogenous insulin production in diabetes
patients). Collagen N-terminal telopeptides are mea-
sured in measured in urine as biomarkers of bone turn-
over (16 ). Thus, body fluids represent attractive
sources to mine for informative proteins and peptides
(Table 2).
Blood fluid (serum or plasma) is regarded as the most
valuable specimen for biomarker elucidation (17 ), be-
cause blood is the transportation medium for most
tissue-derived molecules in the organism. Therefore,
Fig. 2. Generation of a series of peptides from a core
peptide produced via endopeptidase activity.
Amino- and carboxypeptidases can further cleave the core
peptide, generating numerous fragments, which can then
be identified by mass spectrometry
(12, 64)
Table 1. Examples of current peptide biomarkers
used in clinical diagnosis.
Biomarker Fluid Disease
Blood (serum) Heart failure, ventricular
Pro-GRP Blood (serum) Neuroendocrine tumors,
small cell lung cancer
-CTX Blood (serum) Bone turnover
PINP Blood (serum) Bone turnover
Blood (serum) Neuroendocrine tumors
Osteocalcin Blood (serum) Osteoporosis
-Microglobulin Blood (serum) Renal disease and
Calcitonin Blood (serum) Medullary thyroid
Cystatin C Blood (serum) Renal failure
C-peptide Blood (serum),
Diabetes mellitus
VIP Blood (plasma) Pancreatic tumor
ANF Blood (plasma) Heart failure
NTX Urine Bone turnover
-Amyloid (1–42) CSF Alzheimer disease
NT–pro-BNP, N-terminal end of the pro–brain natriuretic peptide; pro-
GRP, pro–gastrin-releasing peptide;
-CTX, cross-linked collagen type I
C-terminal telopeptide; PINP, procollagen type I N-terminal propeptide;
VIP, vasoactive intestinal peptide; ANF, atrial natriuretic factor; NTX,
collagen type 1 N-terminal telopeptide.
Peptidomics in Cancer Reviews
Clinical Chemistry 60:7 (2014) 3
this biofluid can reveal the pathophysiological states of
a broad spectrum of tissues and organs. Compared
with healthy cells, disease-affected cells within tissues
could differentially harbor peptides and proteins even-
tually released into the interstitial fluid and later into
the bloodstream. The high protein content of serum
makes it an attractive fluid for peptidomics; however,
proteins and peptides are present in serum over a wide
and dynamic range of concentrations—10 orders of
magnitude (7 ). This fact represents a considerable an-
alytical challenge, because a few high-abundance pro-
teins (albumin, immunoglobulins, transferrin,
antitrypsin, haptoglobin) hamper the identification of
low-abundance molecules, which are more likely to be
biomarker candidates (18 ). Given that the composi-
tion of blood reflects the metabolic state of the entire
body as it transports molecules released from virtually
any tissue or organ, pathophysiological changes in any
single organ could easily be missed. Because the clot-
ting time has a substantial effect on the polypeptide
content of serum, plasma is often used instead (19 ).
Nevertheless, a few studies have demonstrated the
applicability of serum peptidomic profiling to a range
of medical conditions, although most of these studies
have not been validated for biases and artifacts. Shen et
al. (20 ) explored the plasma peptidome (the entire col-
lection of protein breakdown products) in the quest for
breast cancer biomarkers and detected increased con-
centrations of cancer-relevant protein products, in-
cluding extracellular matrix components, innate
immune system molecules, proteases, and protease in-
hibitors. In another study, Villanueva et al. (13 ) com-
pared the peptidomes of patients with metastatic thy-
roid carcinoma with those of age- and sex-matched
controls and obtained a 12-peptide signature for iden-
tifying malignancy that had a 95% diagnostic sensitiv-
ity and 95% specificity. These and similar approaches
have been challenged for bias and artifacts (http://
Cerebrospinal fluid (CSF) is considered an outstanding
source of biomarkers for neurologic diseases. A color-
less fluid produced in the choroid plexus in the brain,
CSF provides mechanical protection, nutrient supply,
waste product removal, and metabolite transportation.
Via continual interactions, CSF contains molecules
that can reflect many of the processes of the central
nervous system (14 ). Proteomic efforts have been
aimed at characterizing the CSF peptidome to discover
potential biomarkers for neurodegenerative conditions,
neuropsychiatric disorders, traumatic brain injury, brain
tumors, and aging-related conditions (21, 22 ). Wijte et al.
used an enhanced mass spectrometry– based approach
for both free peptides and peptides bound to proteins to
evaluate postmortem CSF from patients with Alzheimer
disease and identified a series of candidate peptides for its
diagnosis, such as VGF, nerve growth factor, and C4 com-
plement precursor (23 ). Additional verification studies
remain to be done.
An analysis by Zougman et al. (14 ) revealed 391
peptides derived from 91 different proteins, and a more
recent study yielded 626 unique peptide sequences of
5 kDa that were derived from 104 proteins (24 ). The
enrichment of CSF with small peptides might be due to
a higher rate of filtration from plasma compared with
components of higher molecular weight.
One of the major disadvantages of CSF studies is the
invasive nature of the collection procedure, making it in-
appropriate for general screening of presumably healthy
individuals or all patients with neuropathologies.
Saliva is a multifunctional body fluid secreted by the
major salivary glands (parotid, submandibular, and
sublingual glands) and other glands distributed in the
oral cavity. It lubricates the oral cavity and participates
in digestion and preventing infections (25 ). Saliva con-
tains bacteria, cellular debris, crevicular fluid, and se-
rum components, and as an alternative sample for
noninvasive collection, saliva has promising, unique
features (26 ). Proteolytic degradation occurs as soon as
proteins enter the oral cavity and continues after a sa-
liva sample is collected. This process leads to great vari-
ation in the peptide profile and thus limits the repro-
ducibility of peptidomic analyses. Other preanalytical
variables, such as sex, age, diet, and circadian rhythms,
can also play important roles in the peptide composi-
tion of saliva (27 ). Despite these shortcomings, pep-
tidomic analyses of saliva have been used to assess a
variety of pathologies, including Sjögren syndrome, xe-
rostomia, and diabetes (28 ). Studies of oral cancer
Table 2. Characteristics of body fluids
for peptidomics.
Body fluid
Invasiveness of
Serum Minimally invasive Unstable
Plasma Minimally invasive Unstable
Urine Noninvasive Stable
CSF Invasive Not known
Saliva Noninvasive Unstable
Ascitic fluid Invasive Unstable
Pleural fluid Invasive Unstable
Tears Noninvasive Not known
4Clinical Chemistry 60:7 (2014)
(29, 30 ) have identified a number of peptides that are
overproduced in patients with squamous cell carci-
noma. Hu et al. (29 ) reported a combination of 5 pro-
teins that could be used to detect oral cancer with 90%
diagnostic sensitivity and 83% specificity.
Tears are a complex extracellular fluid that can be as-
sessed noninvasively. As with plasma, the concentra-
tions of proteins and peptides in tears span several
orders of magnitude (31 ), with low interday but re-
markably pronounced interindividual variation (32 ).
de Souza et al. (33 ) identified 491 proteins in tears, and
a more recent study yielded 1543 proteins (31 ). The
most comprehensive peptidomics study of tears to date
characterized 30 endogenous peptides, most of which
were derived from proline-rich protein 4, a protein of
unknown function produced at high concentrations in
lacrimal acinar cells (34 ). As a body fluid, tears have a
composition that reflects the pathophysiological state
of the underlying tissues and organs and has proved
useful for assessing both ocular and systemic patholo-
gies, such as dry eye, meibomian gland dysfunction,
and Sjögren syndrome (35, 36 ).
Urine as a Source of Biomarkers
Urine is formed in the kidney by ultrafiltration of the
plasma for the elimination of metabolic waste prod-
ucts. Because urine is stored in the bladder for several
hours before elimination, proteolytic degradation by
endogenous proteases has largely been considered to
have been completed by the time of voiding (37 ). Re-
cent findings, however, have demonstrated the pres-
ence of a wide spectrum of proteases in urine (38 ) that
might generate various combinations of different en-
dogenous peptides, depending on how long the urine
resides in the bladder. Proteolytic degradation of a
urine sample can be minimized if second morning
urine samples are used, because the time between the
first and second voiding can be easily monitored and
standardized. Despite such variation, urine is regarded
as stable body fluid, especially compared with blood, in
which proteases are known to be activated during and
after blood drawing and thereby generate a consider-
able number of breakdown products that may skew
proteomic and peptidomic approaches (37, 39 ).
Urine has long been an attractive body fluid for
study, not only in biomarker research but also in clin-
ical diagnosis, mainly because it can be obtained
noninvasively in large amounts. Currently, several
routinely analyzed biomolecules serve as highly dis-
criminating markers. For example, measurements of
urine catecholamines and their metabolites help in as-
sessing pheochromocytoma and in evaluating albu-
minuria as an index of glomerular function.
Some factors require consideration when plan-
ning experiments with urine. Urine shows high daily
biological variation, reflecting the effects that many en-
dogenous and exogenous factors have on its produc-
tion and composition. Diet, exercise, and water intake
are 3 major factors influencing the quality and the
comparability of samples. Urine contains components
released not only from the kidneys and the bladder but
also from many other organs, and biological processes
can have profound impacts on its fluctuating content
(40 ). Even cardiovascular, autoimmune, and infec-
tious diseases affect the presence and concentrations of
some protein molecules (41 ).
Despite containing very small amounts of proteins,
urine samples from healthy and diseased individuals
are attractive for exploring proteomic disease. In 1997,
Heine et al. (42 ) reported the presence of 13 proteins in
human urine. Since then, many other investigators
have assessed the human uroproteome and have drawn
different, methodology-dependent conclusions. The
initial approaches with 2-dimensional electrophoresis
identified 1400 spots (43 ), a number that increased
when liquid chromatography was introduced. Adachi
et al. (44 ) stated that urine from healthy donors con-
tains at least 1543 different proteins, mostly extracellu-
lar and membrane bound. This finding led the authors
to suggest the possibility of specific transport pathways
for lysosomal and plasma membrane proteins for
reaching the urine. The lower protein content of urine
compared with plasma reduces the possibility for high-
abundance proteins to mask potential biomarkers.
Studies have also demonstrated urine to be highly en-
riched for small peptides (45 ); healthy individuals and
patients with Fanconi syndrome contain a 100-fold
enrichment of molecules 10 kDa, compared with
higher molecular weight polypeptides, perhaps be-
cause the former pass freely through the glomerulus
(46 ). On the other hand, low-abundance and low-mass
peptides can become bound to large carrier proteins
that act as harvesters in the circulation (47 ). The major
constituents of the urinary peptidome appear to be col-
lagen fragments, especially from the collagen
1 chain,
which probably reflect the physiological turnover of
tissue extracellular matrix (39 ).
Technological Aspects of Peptidomics
The discovery of novel biomarkers depends not only
on the concentration of the biomarker candidate in the
sample and the complexity of the matrix but also on
the analytical sensitivity of the detection method and
Peptidomics in Cancer Reviews
Clinical Chemistry 60:7 (2014) 5
the sample-preparation steps. The design of study
strategies and analyses of bioinformatics data is crucial
for reproducible and unbiased results (48 ). Any pep-
tidomic analysis requires a robust and comprehensive
To enrich the low molecular weight components in a
sample for peptidomics analyses requires sample-
preparation steps different from those required for
proteomic analyses (17, 49 ). The preanalytical phase is
the most challenging. A wide range of variables, both
exogenous and endogenous, can affect the results
(50, 51 ). The considerable stability of the proteome’s
composition and concentrations in urine allows sam-
ples to be stored for6hatroom temperature with little
change and for years at 20 °C (37, 52 ). Fiedler et al.
investigated the influence of many variables on final
peptidomics results (53 ). Significant differences were
observed not only between first and second morning
urine samples but also between first-stream and mid-
stream urine samples. Bacteriuria and hematuria had a
great effect, even at low concentrations, on the peptide
profile. Freeze–thaw cycles can influence the final re-
sults when assessing exogenous variables; thus, repro-
ducibility is improved with once-frozen urine samples.
To minimize such potentially confounding factors
and preanalytical variations requires that samples be
collected and handled in a standardized manner. The
Human Kidney and Urine Proteome Project (http:// is an international initiative of the
Human Proteome Organization to establish collection
and manipulation procedures for proteomics. In Eu-
rope, the European Kidney and Urine Proteomics or-
ganization ( promotes inter-
actions between scientists in the field, with the goal of
improving the understanding and assessment of kid-
ney disease through urine proteomics. Both associa-
tions have proposed recommendations for standard-
ized urine-processing steps (with special emphasis on
sample collection, centrifugation, and thawing), which
should minimize biases among studies.
Traditionally, hypotheses for biomarker discovery
have been derived from an understanding of disease
biology (54 ). Over the past few decades, however,
many researchers have turned to mass spectrometry to
discover candidate molecules that could serve as bio-
markers. The advantages of mass spectrometry for
identifying and quantifying peptides in complex bio-
logical samples have facilitated the development of
novel biochemical approaches for diagnosis, not only
of cancer but of other diseases as well. Studies of pro-
teins and peptides have used different methodologies.
Two-dimensional gel electrophoresis has been
used extensively, but it is a time-consuming technique
with poor interassay reproducibility, especially at low
molecular weights because it cannot separate and thus
distinguish molecules of 10 kDa. Capillary electro-
phoresis–mass spectrometry yields robust and highly
reproducible analyses of low molecular weight peptides
and is compatible with many volatile buffers and ana-
lytes (19, 22 ); however, the long processing times make
this technique challenging to use for large-scale studies.
One of the most suitable platforms for urine peptide
profiling is SELDI and MALDI followed by mass spec-
trometry identification with a TOF detector. This ap-
proach focuses on peptides in the range of 1–20 kDa.
Immobilization, the key step in the entire process,
reduces sample complexity, but at the expense of a
great loss of information (50 ). Finally, liquid chro-
matography followed by tandem mass spectrometry
(LC-MS/MS) is capable of providing large amounts
of information with high reproducibility. Only cap-
illary electrophoresis and liquid chromatography are
able to interface directly with tandem mass spectrom-
etry instruments for peptidomics studies with the re-
quired depth of analysis, dynamic range, and enhanced
accuracy of quantification (51 ). In addition, analytical
methodologies that increase analytical sensitivity have
been developed. One example is selected reaction mon-
itoring, which uses a nonscanning mode of operation
on an LC-MS/MS instrument (55 ). It increases the de-
tection capability by 2 to 3 orders of magnitude com-
pared with conventional scanning modes.
Mass spectrometry facilitates both biomarker dis-
covery and verification/validation. Mass spectrometers
help in characterizing proteins and peptides and their
modifications. One of the clearest advantages over
other platforms is its capacity to qualitatively screen
and analyze thousands of molecules without previous
knowledge of their existence or relevance to particular
pathophysiological conditions. Quantification of pre-
viously discovered candidates is essential for evaluating
their diagnostic capabilities.
As with proteomics, both absolute and relative
quantification of peptides generally require the use of
stable isotope–labeled molecules as internal standards.
Their use can overcome the problems of matrix effects,
variation in sample preparation, and instrument fluc-
tuations. As outlined elsewhere, the isotopic label
should be introduced into the work flow as early as
possible to increase the number of steps being con-
trolled and thereby decrease imprecision (56 ). This
technique is fairly time-consuming and expensive,
however. Label-free strategies for relative quantifica-
tion are based on comparing signal intensities pro-
duced by identical peptides in different analyses and
rely on the accuracy of the hypothesis that identical
6Clinical Chemistry 60:7 (2014)
peptides will behave similarly across different experi-
ments and therefore permit direct comparisons (57 ).
With urine samples, even absolute quantification
is usually uninformative, so analyte concentration is
commonly corrected for creatinine or protein excre-
tion, or it is based on a 24-h urine collection, thus re-
ducing the dietary and exercise effects on variation in
results. In contrast to transcriptomics and proteomics,
no “housekeeping” peptides have been successfully
identified to date (51 ).
Peptidomics and proteomics require considerable
computing power to obtain statistically significant and
reproducible data. Peptide identification is one of the
most challenging aspects (58 ). Online databases con-
tain peptide sequences for a variety of body fluids and a
myriad of disease conditions, and they serve as univer-
sal platforms for aiding in defining and verifying can-
didate biomarkers (40 ). A substantial proportion of
the human urinary proteome database is derived from
studies that assessed transplantation or renal disease,
whereas the data derived from studies of prostate, re-
nal, and bladder cancers, as well as pheochromocyto-
mas, are relatively few.
The most demanding issue with peptidomics is re-
lated to the nonspecificity of the peptide ends. As
Hölttä et al. have stated (24 ), no restrictions regarding
enzyme-cleavage specificity can be applied during
analyses of bioinformatics data. The consequence is a
huge increase (up to 1000-fold) in the number of se-
quences to consider. This situation contrasts with that
of proteomics, in which protease digestion (usually
with trypsin) ensures specific endings for each peptide
molecule. For this reason, peptidomics suffers from
higher false-positive rates and less accurate results.
Urine Peptidomics for Disease Diagnostics
Most of the literature on urine peptidomics addresses
impairment of kidney function and outcomes of kid-
ney transplantation (41, 59 ). The few studies that have
searched for cancer biomarkers have focused on blad-
der, ovarian, and prostate cancers. Genitourinary ma-
lignancies are responsible for 1 of every 6 cancer deaths
in men and 1 of every 10 in women (1 ).
Ovarian cancer is the deadliest gynecologic malig-
nancy. Current diagnostic strategies are based on mea-
suring carbohydrate antigen 125 (CA125) in serum
(60 ) in combination with vaginal ultrasonography.
Measurement of CA125 lacks diagnostic sensitivity and
specificity for early diagnosis, however, and many ef-
forts have focused on finding protein and peptide mol-
ecules that could be useful as diagnostic biomarkers.
Some proteins, such as human epididymal secretory pro-
tein 4 (61 ) and osteopontin (62 ), have shown utility, al-
though none has surpassed CA125. Using serum, one of
the first peptidomics-based studies combined peaks of
unknown identity, presumably representing low molecu-
lar weight polypeptides, and claimed to distinguish be-
tween individuals with no malignancy and patients with
ovarian cancer (stages I–IV) with 100% sensitivity and
95% specificity (63 ). The results described in this report
have now been invalidated because of preanalytical, ana-
lytical, and bioinformatics artifacts (64 ).
Prostate cancer, the most prevalent malignancy in
men, ranks second in lethality (1 ). Novel noninvasive
markers with higher diagnostic sensitivity and specific-
ity are needed. Although PSA-derived forms and the
ribonucleic acid marker PCA3 (prostate cancer antigen
3) seem to add some degree of diagnostic specificity,
they have not met expectations (65 ). Other protein
candidates that have been suggested require large-scale
validation. The first comparison of the urine proteome
used 2-dimensional gel electrophoresis followed by
MALDI-TOF mass spectrometry fingerprinting of
voided urine samples after prostatic massage to evalu-
ate age-matched men with benign prostatic hyperpla-
sia (66 ). Calgranulin B/MRP-8 was highlighted for ver-
ification and validation. Subsequent research with
urine samples has yielded additional candidate mole-
cules, including the matrix metalloproteinases (67 )
and engrailed-2 (68 ), although none have yet been val-
idated with large cohorts. Hypothesizing that first-void
urine contains prostatic fluid, Theodorescu et al. (69 )
used a filter with a 20-kDa cutoff followed by capillary
electrophoresis–mass spectrometry analysis and ob-
tained a biomarker panel of 12 urinary peptides based
on the results. They proposed that this peptide panel,
used in combination with age, free PSA, and total PSA,
could improve current diagnosis by increasing the area
under the ROC curve from 0.77 (based on the free-PSA
percentage and patient age) up to 0.82. Nevertheless,
this peptide panel also remains to be validated.
Bladder cancer is the fifth most common cancer in
Western societies. Current diagnostic strategies are
based on cytoscopy and urine cytology, but these meth-
ods have high interobserver imprecision and low re-
producibility. Given that the bladder is in intimate
contact with urine after its production in the kidney,
this body fluid has been mined heavily for both protein
and peptide biomarkers that might help, not only in
detecting bladder cancer, but also in distinguishing
muscle-invasive from noninvasive malignancy (70, 71 ).
Peptidomics in Cancer Reviews
Clinical Chemistry 60:7 (2014) 7
A large number of peptides with different concentrations
in urine samples from patients with invasive bladder can-
cer, compared with patients with noninvasive cancer and
with controls, have been found, but most of these peptides
appear to be fragments of abundant proteins. In fact,
Theodorescu et al. (52 ) proposed a proteomic pattern of
22 polypeptides with high diagnostic sensitivity and spec-
ificity for urothelial cancer and highlighted fibrinopeptide
A as a potential diagnostic biomolecule. Bryan et al. (70 )
identified 8 peptides with significantly different concen-
trations in patients with and without muscle-invasive
urothelial carcinoma. Such peptides were identified as
derived from albumin, fibrinogen, hemoglobin, and
prealbumin—all high-abundance proteins.
Anatomically distant sites can influence urine compo-
sition. Studies of the urine peptidome have used this
rationale to pursue possible markers of lung cancer
(72 ) and gastrointestinal cancer (73 ). Using SELDI,
Husi et al. (74 ) found that a nonnegligible number of
the candidate proteins belonged to the family of small
calcium-binding proteins, S100, which have been re-
lated to the growth of tumors of the upper gastrointes-
tinal tract. None of the identified candidates fulfilled
the requirements for a single marker, so a protein–
peptide pattern served for screening and prediction of
outcome. A diagnostic sensitivity of up to 98% was
reported, but most of the candidates had also been de-
scribed for other malignancies, compromising the pat-
tern’s specificity.
If one steps back and considers these results as a
whole, one sees that most of the peptide panels have
not been validated properly. This lack of validation
studies represents one of the major shortcomings of
peptidomics for reaching the clinical setting and places
the usefulness of peptidomics for cancer diagnostics
under a critical eye.
Translation to the Clinic
Despite intensive efforts, no molecule described in any
proteomics or peptidomics study has entered the clinic.
For retrospective and prospective validation studies
of candidate molecules and to avoid artifacts and
methodology-related false-positive results, other meth-
odologies (e.g., immunoassays) are preferred (3, 75 ).
Sometimes initial studies based on small populations
show a statistical significance that, because of bias in
patient selection or other confounders, becomes lost in
subsequent studies. The lessons from this experience
could help in improving the planning of future strate-
gies. Large-scale population studies are rare and carry a
large financial burden. Finally, reaching statistical sig-
nificance is not sufficient for candidate biomarkers. As
with novel drugs, biomarkers have to show some clin-
ical improvement over those currently in use; other-
wise, they will not be adopted.
Given the complexity of any biological process, a
single biomarker has been widely viewed to be unlikely
to discriminate a pathologic process with sufficient
sensitivity and specificity. Therefore, the incorporation
of combinations of multiple, independent biomarkers
into a diagnostic or predictive panel may be more likely
to be useful. Nevertheless, each of the individual bio-
markers used in any panel must be independently ver-
ified and validated to ensure clinical utility. This re-
quirement makes the design of large-scale validation
studies even more difficult.
Recently, a new perspective that transcends classic
proteomics and peptidomics suggests that the study of
individual or global protease activities might also yield
indicators or predictors of disease (76 –78 ). This new
approach has been termed “functional peptidomics.” It
relies on the fact that tumor progression and invasive-
ness may lead to the differential production and secre-
tion of exoproteases; thus, the study of their functions
might not only reflect the true biological/pathologic
state of an organism but also overcome reproducibility
problems related to preanalytical variables. This ap-
proach is still in its beginning stages, however, and con-
clusions about its applicability cannot yet be drawn.
Future Challenges
The impressive growth in high-throughput biology has
dominated science during the last decade, mainly ow-
ing to the leap in the development of new technologies.
Substantial efforts in proteomics have focused on the
discovery and validation of sensitive and specific diag-
nostic biomarkers for many human pathologies. Deep
biochemical and pathophysiological knowledge is crit-
ical for solving clinical questions, and every step in the
procedure must be planned and executed meticu-
lously. Standardized handling procedures are expected
to aid tremendously in the generation of clinically use-
ful and reproducible data.
Peptidomics is a relatively new field, and few studies
of explorations and characterization of the peptidome
have yet been published. There is no strong evidence that
peptidomics will yield better results than proteomics, but
biological and chemical reasoning supports work in that
direction. Proteomics is undoubtedly the dominant tech-
nology in the postgenomics era, and peptidomics repre-
sents a largely unexplored step forward.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3
8Clinical Chemistry 60:7 (2014)
requirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon man-
uscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: E.P. Diamandis, Clinical Chemistry,
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: None declared.
Expert Testimony: None declared.
Patents: None declared.
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10 Clinical Chemistry 60:7 (2014)
... Large-scale research on proteins is termed proteomics, whereas that on naturally occurring peptides is termed peptidomics [9,10]. High-resolution mass spectrometry (MS), especially data-independent acquisition MS (DIA-MS), has enabled broad application of proteomics in medical science. ...
... However, the research on understanding the role of proteases and peptidomics in IMN is still very limited. Proteases are necessary to maintain tissue homeostasis, and their dysregulation may be related to the production of urinary peptides in CKD [3,9,10]. Proteasix, a bioinformatics tool, is dedicated to exploring proteases that produce peptides. ...
Full-text available
Background Idiopathic membranous nephropathy (IMN) is a cause of nephrotic syndrome that is increasing in incidence but has unclear pathogenesis. Urinary peptidomics is a promising technology for elucidating molecular mechanisms underlying diseases. Dysregulation of the proteolytic system is implicated in various diseases. Here, we aimed to conduct urinary peptidomics to identify IMN-related proteases. Results Peptide fingerprints indicated differences in naturally produced urinary peptide components among 20 healthy individuals, 22 patients with IMN, and 15 patients with other kidney diseases. In total, 1,080 peptide-matched proteins were identified, 279 proteins differentially expressed in the urine of IMN patients were screened, and 32 proteases were predicted; 55 of the matched proteins were also differentially expressed in the kidney tissues of IMN patients, and these were mainly involved in the regulation of proteasome-, lysosome-, and actin cytoskeleton-related signaling pathways. The 32 predicted proteases showed abnormal expression in the glomeruli of IMN patients based on Gene Expression Omnibus databases. Western blot revealed abnormal expression of calpain, matrix metalloproteinase 14, and cathepsin S in kidney tissues of patients with IMN. Conclusions This work shown the calpain/matrix metalloproteinase/cathepsin axis might be dysregulated in IMN. Our study is the first to systematically explore the role of proteases in IMN by urinary peptidomics, which are expected to facilitate discovery of better biomarkers for IMN.
... These bodily fluids are the liquids that remain in the body. These consist of pleural fluids, blood plasma, blood serum, cerebrospinal fluid, and ascitic fluid [156]. Other applications of invasive body fluids include glucose monitoring [157], proteome analysis [158], the creation of wearable electrochemically active biosensors [159], the detection of antibodies [160], postmortem toxicology profiles, the diagnosis of Alzheimer's disease using specific peptides [161], and the identification of biomarkers for various diseases. ...
A wide range of applications are possible with paper-based analytical devices, which are low priced, easy to fabricate and operate, and require no specialized equipment. Paper-based microfluidics offers the design of miniaturized POC devices to be applied in the health, environment, food, and energy sector employing the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment free and Deliverable to end users) principle of WHO. Therefore, this field is growing very rapidly and ample research is being done. This review focuses on fabrication and detection techniques reported to date. Additionally, this review emphasises on the application of this technology in the area of medical diagnosis, energy generation, environmental monitoring, and food quality control. This review also presents the theoretical analysis of fluid flow in porous media for the efficient handling and control of fluids. The limitations of PAD have also been discussed with an emphasis to concern on the transformation of such devices from laboratory to the consumer.
... Notwithstanding the participation of saliva in early phases of food digestion, very few peptidomic studies have been performed so far on food-derived peptides in this biological fluid; the limited examples in this context are associated with recent studies on the generation of bitter-and salty-taste associated peptides in this fluid (Stolle et al., 2018;Sebald et al., 2020). Such a lack is probably the result of the complexity and variability of the salivary peptidome as related to gender, age, and circadian/seasonal rhythms (Bauça et al., 2014). Similarly, only very few peptidomic studies were performed on food-derived peptides in animal tissues/biopsies, namely antioxidant glutathione in liver (Yamada et al., 2018), antihypertensive VY in liver, kidney, heart and lung (Matsui et al., 2004;Tanaka et al., 2015), and adiponectin-receptor agonist YP in brain tissues (Tanaka et al., 2019;Lee et al., 2021). ...
Background Various bioactive peptides are present in foods and food protein hydrolysates, or are generated in the stomach/intestine of organisms after digestion of dietary proteins. Those resisting gastrointestinal degradation can exert local effects in the gut or systemic effects in the organism body as result of their transport across the intestinal epithelium in the bloodstream, and subsequent adsorption in various organs. For most of these molecules, no concentration data regarding body fluids/tissues are available; this information is essential to rationalize their bioavailability and putative bioactivity. Scope and approach The main purpose of this study is to provide an exhaustive overview of the bioactive food-derived peptides identified in the gastrointestinal tract, blood, body tissues, urine, breastmilk and feces of animal models or humans fed specific diets, as well as a description of the adsorption mechanisms and metabolic processes eventually affecting their fate. Untargeted and targeted peptidomic methods used for their quali-quantitative description are also reported, together with recent technological advances that have partially solved various analytical challenges in this research field and have disclosed future promising scenarios in nutrition and physiology. Key findings and conclusions Available information emphasizes that organism tissues/body fluids are pervaded of food-derived species resulting from the digestion of dietary proteins, including some already proved having a specific biological activity. For some for which blood concentration was measured, ascertained data highlight levels in the nanomolar range, which are lower than those generally used for in vitro functional assays. Conversely, few peptides have shown concentration values compatible with a substantial molecular bioavailability and a putative bioactivity. Thus, it remains uncertain if the presence of bioactive food-derived peptides in the body fluids/tissues can be associated with a significant functional effect. Accordingly, the actual study of these exogenous peptides in the human body is more relevant than ever, with the ultimate aim of tangling the complex relationship between diet and health.
... Human urine is one of the most interesting biofluids for clinical proteomic studies, advances in MS leading to the identification of thousands of proteins and peptides in urine [402]. Urine peptidomics and uroproteome alterations study represent alternative approaches for discovering candidate biomarkers, especially in early cancer diagnosis [403], discriminating between breast cancer patients from healthy controls [404]. Urinary zinc-alpha2-glycoprotein (ZA2G), leucine-rich alpha2-glycoprotein (LRG), retinolbinding protein A4 (RBP4), also emphasizing significant higher serum levels in patients with breast cancer [405], annexin A1 (ANXA1), ganglioside GM2 activator (SAP3/GM2A), Src substrate cortactin (SRC8/CTTN), gelsolin (GELS/ GSN), kininogen-1 (KNG1), CO9, clusterin (CL-US) that when overexpressed might be a predictive factor for recurrence [406], whereas ceruloplasmin (CERU), and α1-antitrypsin (A1-AT) have been proposed as candidate biomarkers that could discriminate HER2-enriched subtype breast cancer from the healthy controls [404]. ...
Breast cancer is an individually unique, multi-faceted and chameleonic disease, an eternal challenge for the new era of high-integrated precision diagnostic and personalized oncomedicine. Besides traditional single-omics fields (such as genomics, epigenomics, transcriptomics and metabolomics) and multi-omics contributions (proteogenomics, proteotranscriptomics or reproductomics), several new "-omics" approaches and exciting proteomics subfields are contributing to basic and advanced understanding of these "multiple diseases termed breast cancer": phenomics/cellomics, connectomics and interactomics, secretomics, matrisomics, exosomics, angiomics, chaperomics and epichaperomics, phosphoproteomics, ubiquitinomics, metalloproteomics, terminomics, degradomics and metadegradomics, adhesomics, stressomics, microbiomics, immunomics, salivaomics, materiomics and other biomics. Throughout the extremely complex neoplastic process, a Breast Cancer Cell Continuum Concept (BCCCC) has been modeled in this review as a spatio-temporal and holistic approach, as long as the breast cancer represents a complex cascade comprising successively integrated populations of heterogeneous tumor and cancer-associated cells, that reflect the carcinoma's progression from a "driving mutation" and formation of the breast primary tumor, toward the distant secondary tumors in different tissues and organs, via circulating tumor cell populations. This BCCCC is widely sustained by a Breast Cancer Proteomic Continuum Concept (BCPCC), where each phenotype of neoplastic and tumor-associated cells is characterized by a changing and adaptive proteomic profile detected in solid and liquid minimal invasive biopsies by complex proteomics approaches. Such a profile is created, beginning with the proteomic landscape of different neoplastic cell populations and cancer-associated cells, followed by subsequent analysis of protein biomarkers involved in epithelial-mesenchymal transition and intravasation, circulating tumor cell proteomics, and, finally, by protein biomarkers that highlight the extravasation and distant metastatic invasion. Proteomics technologies are producing important data in breast cancer diagnostic, prognostic, and predictive biomarkers discovery and validation, are detecting genetic aberrations at the proteome level, describing functional and regulatory pathways and emphasizing specific protein and peptide profiles in human tissues, biological fluids, cell lines and animal models. Also, proteomics can identify different breast cancer subtypes and specific protein and proteoform expression, can assess the efficacy of cancer therapies at cellular and tissular level and can even identify new therapeutic target proteins in clinical studies.
... The fluids found inside the human body are known as invasive body fluids. These include blood plasma, blood serum, cerebrospinal fluid, ascitic fluid and pleural fluids [49]. These have also been used for glucose monitoring [50] proteome analysis [51], development of electrochemically active biosensors which can be worn [52], detection of antibodies [53], postmortem toxicology profiles [54], diagnosis of Alzheimer's disease using certain peptides [55], detection of biomarkers for various diseases [56]. ...
Paper has been used for testing analytes since the advent of litmus paper. Also, rapid growth of the human population in isolated areas of the world have engendered demand for testing and diagnostic methods that are not only easy to transport, but also have a low cost of fabrication and rapid results. Hormones are crucial biomarkers which can be used to detect certain physiological conditions. These hormones, present in non-invasive and invasive body fluids, hold major potential for detection using paper-based devices. This review paper aims to highlight the advancements in creation of paper-based microfluidic devices to detect hormones in different body fluids. It exhaustively explains the current methods of detection for hormones in various body fluids like sweat, saliva and serum using paper-based microfluidic devices and the role of these devices in the future in terms of rapid detection, low cost, and ease of transport. By demonstrating the mechanisms of these devices, this paper shows a promising unconventional avenue for efficient hormone detection.
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Peptidomics allows the identification of peptides that are derived from proteins. Urinary peptidomics has revolutionized the field of diagnostics as the samples represent complete systemic changes happening in the body. Moreover, it can be collected in a non-invasive manner. We profiled the peptides in urine collected from different physiological states (heifer, pregnancy, and lactation) of Sahiwal cows. Endogenous peptides were extracted from 30 individual cows belonging to three groups, each group comprising of ten animals (biological replicates n = 10). Nano Liquid chromatography Mass spectrometry (nLC-MS/MS) experiments revealed 5239, 4774, and 5466 peptides in the heifer, pregnant and lactating animals respectively. Urinary peptides of <10 kDa size were considered for the study. Peptides were extracted by 10 kDa MWCO filter. Sequences were identified by scanning the MS spectra ranging from 200 to 2200 m/z. The peptides exhibited diversity in sequences across different physiological states and in-silico experiments were conducted to classify the bioactive peptides into anti-microbial, anti-inflammatory, anti-hypertensive, and anti-cancerous groups. We have validated the antimicrobial effect of urinary peptides on Staphylococcus aureus and Escherichia coli under an in-vitro experimental set up. The origin of these peptides was traced back to certain proteases viz. MMPs, KLKs, CASPs, ADAMs etc. which were found responsible for the physiology-specific peptide signature of urine. Proteins involved in extracellular matrix structural constituent (GO:0005201) were found significant during pregnancy and lactation in which tissue remodeling is extensive. Collagen trimers were prominent molecules under cellular component category during lactation. Homophilic cell adhesion was found to be an important biological process involved in embryo attachment during pregnancy. The in-silico study also highlighted the enrichment of progenitor proteins on specific chromosomes and their relative expression in context to specific physiology. The urinary peptides, precursor proteins, and proteases identified in the study offers a base line information in healthy cows which can be utilized in biomarker discovery research for several pathophysiological studies. Excretory biological fluids such as urine, saliva, milk, tear, mucus and sweat are significantly important for the maintenance of homeostasis in normal physiological conditions. Urine being a glomerular filtrate of blood is capable of summarizing the events that occur in the body as a result of changing physiology or pathological conditions. The systemic changes are well reflected by qualitative and quantitative alterations in the urine composition. No wonder, urine has been considered as excellent sample for the discovery and detection of biomarkers associated with general health and disease. Endogenous peptides and proteins secreted in urine have proven as hallmarks of various pathophysiological changes and have emerged as a better option over other biological fluids as it can be obtained in a large volume without much maneuver in dairy animals. The quantitative estimation of urinary biomarkers is affected by changes in the volume of voided urine 1. In the absence of a standard baseline profile of peptides in the urine, identification and quantitation of biomarkers OPEN
Biomedicine is developing rapidly in the 21st century. Among them, the qualitative and quantitative analysis of peptide biomarkers is of considerable importance for the diagnosis and therapy of diseases and the quality evaluation of drugs and food. The identification and quantitative analysis of peptides have been going on for decades. Traditionally, immunoassays or biological assays are generally used to quantify peptides in biological matrices. However, the selectivity and sensitivity of these methods cannot meet the requirements of the application. The separation and analysis technique of liquid chromatography‐mass spectrometry (LC–MS) supplies a reliable alternative. In contrast to immunoassays, LC–MS methods are capable of providing the analytical prowess necessary to satisfy the demands of peptide biomarker research in the life sciences arena. This review article provides a historical account of the in‐roads made by LC–MS technology for the detection of peptide biomarkers in the past 10 years, with the focus on the qualification/quantification developments and their applications.
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Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating pain disorder of the bladder and urinary tract with poorly understood etiology. A definitive diagnosis of IC/BPS can be challenging because many symptoms are shared with other urological disorders. An analysis of urine presents an attractive and non-invasive resource for monitoring and diagnosing IC/BPS. The antiproliferative factor (APF) peptide has been previously identified in the urine of IC/BPS patients and is a proposed biomarker for the disorder. Nevertheless, other small urinary peptides have remained uninvestigated in IC/BPS primarily because protein biomarker discovery efforts employ protocols that remove small endogenous peptides. The purpose of this study is to investigate the profile of endogenous peptides in IC/BPS patient urine, with the goal of identifying putative peptide biomarkers. Here, a non-targeted peptidomics analysis of urine samples collected from IC/BPS patients were compared to urine samples from asymptomatic controls. Our results show a general increase in the abundance of urinary peptides in IC/BPS patients, which is consistent with an increase in inflammation and protease activity characteristic of this disorder. In total, 71 peptides generated from 39 different proteins were found to be significantly altered in IC/BPS. Five urinary peptides with high variable importance in projection (VIP) coefficients were found to reliably differentiate IC/BPS from healthy controls by receiver operating characteristic (ROC) analysis. In parallel, we also developed a targeted multiple reaction monitoring method to quantify the relative abundance of the APF peptide from patient urine samples. Although the APF peptide was found in moderately higher abundance in IC/BPS relative to control urine, our results show that the APF peptide was inconsistently present in urine, suggesting that its utility as a sole biomarker of IC/BPS may be limited. Overall, our results revealed new insights into the profile of urinary peptides in IC/BPS that will aid in future biomarker discovery and validation efforts.
Method: In the context of precision medicine, disease treatment requires individualized strategies based on the underlying molecular characteristics to overcome therapeutic challenges posed by heterogeneity. For this purpose, it is essential to develop new biomarkers to diagnose, stratify, or possibly prevent diseases. Plasma is an available source of biomarkers that greatly reflects the physiological and pathological conditions of the body. An increasing number of studies are focusing on proteins and peptides, including many involving the Human Proteome Project (HPP) of the Human Proteome Organization (HUPO), and proteomics and peptidomics techniques are emerging as critical tools for developing novel precision medicine preventative measures. Excitingly, the emerging plasma proteomics and peptidomics toolbox exhibits a huge potential for studying pathogenesis of diseases (e.g., COVID-19 and cancer), identifying valuable biomarkers and improving clinical management. However, the enormous complexity and wide dynamic range of plasma proteins makes plasma proteome profiling challenging. Herein, we summarize the recent advances in plasma proteomics and peptidomics with a focus on their emerging roles in COVID-19 and cancer research, aiming to emphasize the significance of plasma proteomics and peptidomics in clinical applications and precision medicine. This article is protected by copyright. All rights reserved.
Background Bladder cancer (BC) is one of the ten most common types of cancer worldwide, with approximately 550,000 new cases each year. Early detection and appropriate diagnosis are important factors in successful treatment of the disease. Materials and Methods We used specific fluorogenic substrate for the quantitative determination of urine kallikrein 13 (KLK13) activity in healthy (n = 15) and bladder cancer (n = 54) patients. The proteolytic activity in individual urine samples was determined by fluorescence measurements. Then immunoenzymatic analyzes (ELISA, Western blot) were performed to confirm the presence of KLK13 in the tested samples. Results Urine samples from patients with G2 and G3 grade bladder cancer contained proteolytically active KLK13, as confirmed by kinetic analysis and immunochemical detection. KLK13 was not detected in the urine of patients with G1 grade bladder cancer. Discussion Previous clinical study reveal the KLK13 significance for bladder cancer prognosis as increased KLK13 expression was highlighted in bladder tumors compared to normal adjacent tissues. Our findings correlates to the report. KLK13 activity was confirmed in bladder cancer patients with G2 and G3 stage of disease development. Conclusion Using specific chromogenic/fluorogenic peptides could be useful for the noninvasive disease monitoring of bladder cancer progress.
Context Development of new biomarkers for ovarian cancer is needed for early detection and disease monitoring. Analyses involving complementary DNA (cDNA) microarray data can be used to identify up-regulated genes in cancer cells, whose products may then be further validated as potential biomarkers. Objective To describe validation studies of an up-regulated gene known as osteopontin, previously identified using a cDNA microarray system. Design, Setting, and Participants Experimental and cross-sectional studies were conducted involving ovarian cancer and healthy human ovarian surface epithelial cell lines and cultures, archival paraffin-embedded ovarian tissue collected between June 1992 and June 2001, and fresh tissue and preoperative plasma from 144 patients evaluated for a pelvic mass between June 1992 and June 2001 in gynecologic oncology services at 2 US academic institutions. Plasma samples from 107 women selected from an epidemiologic study of ovarian cancer initiated between May 1992 and March 1997 were used as healthy controls. Main Outcome Measures Relative messenger RNA expression in cancer cells and fresh ovarian tissue, measured by real-time polymerase chain reaction as 2−ΔΔCT(a quantitative value representing the amount of osteopontin expression); osteopontin production, localized and scored in ovarian healthy and tumor tissue with immunohistochemical studies; and amount of osteopontin in patient vs control plasma, measured using an enzyme-linked immunoassay. Results The geometric mean for 2−ΔΔCTfor osteopontin expression in 5 healthy ovarian epithelial cell cultures was 4.1 compared with 270.4 in 14 ovarian cancer cell lines (P = .03). The geometric mean 2−ΔΔCTfor osteopontin expression in tissue from 2 healthy ovarian epithelial samples was 9.0 compared with 164.0 in 27 microdissected ovarian tumor tissue samples (P = .06). Immunolocalization of osteopontin showed that tissue samples from 61 patients with invasive ovarian cancer and 29 patients with borderline ovarian tumors expressed higher levels of osteopontin than tissue samples from 6 patients with benign tumors and samples of healthy ovarian epithelium from 3 patients (P = .03). Osteopontin levels in plasma were significantly higher (P<.001) in 51 patients with epithelial ovarian cancer (486.5 ng/mL) compared with those of 107 healthy controls (147.1 ng/mL), 46 patients with benign ovarian disease (254.4 ng/mL), and 47 patients with other gynecologic cancers (260.9 ng/mL). Conclusions Our findings provide evidence for an association between levels of a biomarker, osteopontin, and ovarian cancer and suggest that future research assessing its clinical usefulness would be worthwhile.
The field of proteomics is undergoing rapid development in a number of different areas including improvements in mass spectrometric platforms, peptide identification algorithms and bioinformatics. In particular, new and/or improved approaches have established robust methods that not only allow for in-depth and accurate peptide and protein identification and modification, but also allow for sensitive measurement of relative or absolute quantitation. These methods are beginning to be applied to the area of neuroproteomics, but the central nervous system poses many specific challenges in terms of quantitative proteomics, given the large number of different neuronal cell types that are intermixed and that exhibit distinct patterns of gene and protein expression. This review highlights the recent advances that have been made in quantitative neuroproteomics, with a focus on work published over the last five years that applies emerging methods to normal brain function as well as to various neuropsychiatric disorders including schizophrenia and drug addiction as well as of neurodegenerative diseases including Parkinson's disease and Alzheimer's disease. While older methods such as two-dimensional polyacrylamide electrophoresis continued to be used, a variety of more in-depth MS-based approaches including both label (ICAT, iTRAQ, TMT, SILAC, SILAM), label-free (label-free, MRM, SWATH) and absolute quantification methods, are rapidly being applied to neurobiological investigations of normal and diseased brain tissue as well as of cerebrospinal fluid (CSF). While the biological implications of many of these studies remain to be clearly established, that there is a clear need for standardization of experimental design and data analysis, and that the analysis of protein changes in specific neuronal cell types in the central nervous system remains a serious challenge, it appears that the quality and depth of the more recent quantitative proteomics studies is beginning to shed light on a number of aspects of neuroscience that relates to normal brain function as well as of the changes in protein expression and regulation that occurs in neuropsychiatric and neurodegenerative disorders.
Objective: To assess and characterize the temporal variation in ovarian cancer incidence and mortality by age within countries in the Americas, Europe, Asia, and Oceania. Methods/materials: Data from the National Cancer Institute's Surveillance, Epidemiology, and End Results Program in the United States (U.S.) were used to assess ovarian cancer incidence rates (1998-2008) and mortality rates, (1988-2007 for 12-month survival, 1988-2006 for 24-month survival, and 1988-2003 for 60-month survival), stratified by age at diagnosis. Data from GLOBOCAN were used to calculate country-specific incidence rates for 2010 and 2020 and case-fatality rates for 2010. Results: A statistically significant decrease in Annual Percent Change (APC) of ovarian cancer incidence was observed in the U.S. for all women (-1.03%), among women who were diagnosed at <65 years of age (-1.09%) and among women who were diagnosed at ≥65 years of age (-0.95%). There was a statistically significant increase in the observed APC for survival at 12-months (0.19%), 24-months (0.58%), and 60-months (0.72%) for all women; however, 5-year survival for advanced stage (III or IV) disease was low at less than 50% for women <65 years and less than 30% for women ≥65 years. Global results showed a wide range in ovarian cancer incidence rates, with China exhibiting the lowest rates and the Russian Federation and the United Kingdom exhibiting the highest rates. Conclusions: Ovarian cancer survival has shown modest improvement from a statistical perspective in the U.S. However, it is difficult to ascertain how clinically relevant these improvements are at the population or patient level.