Proteomics: Biomedical and Pharmaceutical Applications || Proteomics and Peptidomics of Gestational...

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Chapter 8

Proteomics and Peptidomics of Gestational Disease

NIGEL M. PAGE School of Animal and Microbial Sciences, The University of Reading, Reading, RG6 6AJ, UK

1. INTRODUCTION

Prenatal screening has become an important and routine part of modern

obstetric care and the necessity for developing novel diagnostic markers

remains a challenging one. The demand for prenatal screening is continually

growing and it is estimated that approximately two thirds of pregnant women

in the United States are having placental marker serum screening for

conditions such as Down’s syndrome (DS) (Palomaki et al., 1997). Indeed,

the first test a women will encounter to confirm her pregnancy is based on

the detection of the α and β polypeptide subunits of human chorionic

gonadotrophin (hCG) in her urine, hCG being the most established of the

placental peptide markers. The advantages of this test are that not only is

hCG secreted by the placenta and specific to pregnancy (except in some

forms of cancer such as choriocarcinoma and testicular cancer) but levels of

hCG rise exponentially in the first few weeks. Unfortunately, such clear-cut

diagnosis is not at present always possible for gestational disease, as

placental markers are not necessarily exclusive to the condition being

screened for; rather they simply enable an estimation of risk.

This chapter introduces the major gestational diseases in respect to the

development of prenatal screening describing the developments from the

preproteomics era to current research and to the future role of proteomics in

the discovery of novel placental peptide markers. Here, the additional

proteomic challenges that are needed to accommodate the study of peptides,

being that, by their nature they are smaller and consequently have different

© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

H. Hondermarck (ed.), Proteomics: Biomedical and Pharmaceutical Applications, 183–199.

184 Nigel M. Page et al.

physical chemistry properties from those of proteins are addressed. Hence,

the developing field of peptidomics is introduced. It is thus hoped that the

application of peptidomics will lead to the design of robust, rapid and clinic

friendly diagnostic assays.

2. THE PLACENTA AND GESTATIONAL DISEASE

The development of the foetus within the maternal uterus is linked

anatomically, endocrinologically and metabolically with that of the placenta.

This temporary structure with its forty week life span is unique to pregnancy

taking on the role of a multiple organ system. It is derived from the outer

trophectoderm cells of the blastocyst, which establish physical contact with

the maternal uterine endometrium at implantation giving rise to the

trophoblast. It is the proliferating cells of the trophoblast that invade and

destroy adjacent decidua cells of the endometrium. A process that leads to

the general anatomical layout of the placenta being complete within the first

three to four weeks of pregnancy. A full blood flow is established at around

10 to 12 weeks - a period that represents a change in the placental

environment from relative hypoxia to an increase in oxygen tension (Jaffe et

al. 1997). Optimal placental perfusion requires the controlled invasion of the

trophoblast cells deep into the myometrial spiral arteries; whereby the

narrow walls of the spiral arteries are replaced with the trophoblasts,

rendering them flaccid and distended, and resulting in an increase of blood

flow to the placenta. In the mature placenta, fingers of villi derived from the

trophoblast dip into maternal blood spaces providing a large surface area for

exchange. This provides nourishment and oxygen for the developing foetus

and placenta and allows an ideal place for proteins/peptides to be secreted

across the foetal-maternal boundary. Pathologies of the placenta and factors

affecting its development play a key role in establishing gestational disease.

We, therefore, take a look at the pathological diversities within the major

gestational diseases.

1.1 Foetal growth retardation

Foetal growth retardation (FGR) is defined as a predicted foetal weight

at term that is under the 10th percentile or below 2.5 kg. It is linked to

significantly elevated foetal morbidity and mortality (McCormick, 1985),

with the most common causes cited as chronic hypertension, pre-eclampsia,

smoking, alcohol, stress and intrauterine infections. It is believed that these

conditions cause abnormalities in placental structure and function (Regnault

et al., 2002) interfering with implantation, placentation, placental

Proteomics and peptidomics of gestational diseases 185

metabolism and transport (Pardi et al., 2002). A plethora of growth factors

and hormones such as insulin-like growth factors (IGF), fibroblast growth

factors (FGF), epidermal growth factors (EGF), transforming growth factors

(TGF) and platelet derived growth factors (PDGF) have all been implicated.

Many of which exhibit altered expression within the placenta of FGR

pregnancies.

2.1 Pre-term labour

Pre-term birth is a major contributor to perinatal mortality and morbidity

and in the USA approximately 11% of births occur before the 37th week

(Andrews et al., 2000). Moreover, the actual pre-term birth rate has

increased over the last 30 years (Mauldin & Newman, 2001), although

advances in neonatal care have resulted in a significant increase in successful

outcomes. The regulatory pathways leading to parturition in humans are not

well defined and appear to be multifactorial. These include the interleukins,

endothelins, oxytocin, urocortin and various steroid hormones.

Corticotrophin releasing factor (CRF) has been postulated to be the peptide

that sets in motion the positive feedback loops that results in parturition

(McLean & Smith, 1999), being released by the placenta in exponentially

increasing amounts during pregnancy (Campbell et al., 1987). Leading

theories for pre-term birth include infections, inflammatory and ischaemic

damage to the placenta (Mauldin & Newman, 2001). Aside from the major

economic costs associated with caring for pre-term neonates,

epidemiological evidence suggests that there is an increased risk of disease

in later life including heart disease, type-2 diabetes and hypertension

(Barker, 1989).

2.2 Pre-eclampsia

Pre-eclampsia (PE) affecting 3-10% of pregnancies, is a principal cause

of maternal morbidity and mortality accounting for almost 15% of

pregnancy-associated deaths and is a major cause of iatrogenic prematurity

among new borns. Mild PE includes increases in maternal blood pressure

and proteinuria (NIH, 2000) and can develop unpredictably into severe PE

over a matter of days or weeks. There is a vast diversity of symptoms

associated with severe PE including cerebral oedema, neurological

manifestations, liver capsule distension, renal failure, pulmonary oedema

and thrombocytopenia (Page, 2002). A defective trophoblastic invasion of

the placental bed is thought to result in hypoperfusion and an ischaemic

placenta, with the release of unknown factors into the maternal circulation.

Markers observed in PE are the powerful vasoconstrictors (e.g. endothelins

and angiotensins) and those that compensate for these hypertensive effects


(e.g. atrial natriuretic factor, vasoactive intestinal polypeptide,

adrenomedullin). There are also a number of non-vasoactive peptides

implicated (e.g. leptin, β-hCG, inhibin-A).

2.3 Chromosomal Disorders

Genomic changes, such as aneuploidy, deletions and other chromosomal

rearrangements, have long been associated with pregnancy loss and

congenital abnormalities. Typically, these are errors in recombination, and

therefore cannot often be predicted prior to fertilisation, though certain risk

factors, such as increasing maternal age, indicate a greater risk. The most

common chromosomal disorder is DS (trisomy 21), a congenital syndrome

with a median birth incidence of 1 per 1000 births with rates increased to 33

per 1000 at the age of 45 years (Hook, 1981). The syndrome includes

hypotonia, mental and growth retardation, heart defects and an increased

incidence of leukemia and Alzheimers disease with associated high rates of

infant mortality (Mikkelsen et al., 1990). Other examples of chromosomal

disorders include Patau syndrome (trisomy 13), Edwards syndrome (trisomy

18), Turner syndrome (X0), Klinefelter syndrome (XXY), Cri-du-chat

syndrome (deleted 5p) and Prader-Willi syndrome (lack of the paternal copy

of chromosome 15q). Abnormal serum levels of α-fetoprotein, hCG, inhibin-

A and unconjugated oestriol are all associated with DS (Wald et al., 1996).

2.4 Gestational Diabetes

Diabetes mellitus is a common complication of pregnancy affecting 2-

15% of pregnancies, of which 90% are classified as gestational diabetes

mellitus (Tamas & Kerenyi, 2001). Unlike women with type 1 diabetes,

women with gestational diabetes have plenty of insulin. However, the effect

of their insulin is partially blocked by a variety of hormones secreted by the

placenta, such as oestrogen, cortisol and human placental lactogen. Insulin

resistance usually begins about week 20 of pregnancy and increases with

placental development. A major problem affiliated with gestational diabetes

is macrosomia, a considerably larger than normal baby. This occurs when

the transport of nutrients such as glucose across the utero-placental unit

becomes unregulated resulting in high glucose levels in the foetal and

maternal plasma. Leptin is also elevated in infants with type 1 diabetic and

gestational diabetic mothers (Persson et al., 1999), while IGF-I, IGF-II and

other growth factors including the FGFs are increasingly expressed in such

placental tissue (Arnay and Hill, 1998, Hill et al., 1998).


2.5 Trophoblastic Disease

Trophoblastic disease includes gestational trophoblastic disease, a

spectrum of rare neoplastic conditions, and gestational trophoblastic tumours

(GTTs), the abnormal proliferation of different types of trophoblasts. These

diseases vary from partial hydatidiform mole to choriocarcinoma. GTTs are

always histologically choriocarcinoma and secrete the β-hCG more

abundantly than normal. The serum or urinary level of this subunit is

proportional to the tumour volume and represents a fundamental basis for

follow-up of these placental tumours (Elegbe et al., 1984). EGF expression

is also found to be higher in molar placenta of all gestational ages, linking its

role to the proliferative and differentiating activity of the trophoblast (John et

al., 1997).

3. OUTCOMES FROM THE PREPROTEOMICS

ERA

The first characterisation of a peptide in pregnancy and its role in

diagnosis was for hCG by Ascheim (1927). This utilised a bioassay that was

designed to observe the enlargement and luteinisation of the corpus luteum

of the mouse following injections of urine from pregnant women. Later

bioassays applying agglutination methods helped to improve and simplify

hCG testing, however, sensitivities in pregnancy diagnosis were not seen till

the 1960s when immunoassays were developed (Yagami & Ito, 1965) and

with the advent of monoclonal antibodies (Wahlstrom et al., 1981).

However, no test, actually identified a gestational abnormality using a

protein or peptide until 1972, when it was first reported that maternal serum

α-fetoprotein (AFP) levels were raised in foetuses affected by neural tube

defect (Brock & Sutcliffe, 1972), a defect that can lead to spina bifida. AFP,

a uniquely foetal protein produced by the liver was found to enter the

maternal circulation with a linear relationship to week 20 of pregnancy,

where higher than normal levels were found to indicate leaking protein from

an open neural tube. A year later, raised maternal serum levels of AFP were

found to be associated with anencephaly (Brock et al., 1973) and in 1977 the

first prenatal screening programme for birth defects was established (Wald et

al., 1977). In 1983, Merkatz et al. conducted a study of 53 pregnancies

affected by chromosomal abnormalities. This followed the observation that a

mother who gave birth to a child with trisomy 18 was found to have

consistently low levels of AFP throughout pregnancy. They discovered that

43 of the affected cases had serum AFP levels below the mean value for

unaffected pregnancies. A wider study taken from pregnancies affected with


trisomy 21 found mean AFP values were approximately 25% lower than in

unaffected pregnancies (Cuckle et al., 1984). This lead to a method that by

also taking into account the maternal age found to be an independent

variable, was able to predict 35% of DS pregnancies with a 5% false-positive

rate (Cuckle et al., 1987). hCG levels were found to be about twice as high

in DS pregnancies (Bogart et al., 1987). This lead to the establishment of

multiple screening tests to establish an estimation of risk. In the case of DS

screening AFP, hCG and unconjugated oestriol levels (found to be lower in

DS pregnancies) became known as the ‘triple test’ (Wald et al., 1994) and

later the ‘quadruple test’ with the inclusion of inhibin-A (Wald et al., 1996).

Much of the history of the preproteomic era has been dominated by similar

examples of fortuitous placental peptide/protein isolation, the raising of

specific antisera and the controlled matching and screening of placental

extracts and maternal serum from both normal pregnancies and those

affected by gestational disease. This process more often than not being

driven by serendipity and by the availability of patient samples to individual

investigators.

4. PROTEOMICS TO DATE

It is now possible to abandon serendipity and utilise modern proteomic

approaches to search for novel markers of pregnancy that may be useful in

diagnosis and screening. However, the use of proteomic approaches has yet

to see widespread acceptance and incorporation into the field of obstetrics.

To date there are only a handful of studies relating to gestational disease

applying proteomics. An extensive PubMed search has revealed only four

experimental papers so far published on the proteomics of the placenta of

which only two relate to gestational disease and two reviews (Page et al.,

2002, Romero et al., 2002). In the first paper Rabilloud et al. (2001) report

the construction of a human mitochondrial proteome using placenta as the

source material. They used two-dimensional (2-D) electrophoresis and

peptide mass fingerprinting in an attempt to build a picture of this

organelle’s proteome. Their approach though not directly aimed at any

particular disease could provide interesting reference data. Mitochondria are

inherited through the mother and have been shown to play a role in many

non-Mendelian inherited diseases (Cummins, 2002). Mitochondria are also

an important source of oxidative stress and lipid peroxidation increased

activities of which are associated with PE. Not only this, but the number of

placental mitochondria are increased during PE (Wang & Walsh, 1998).

Hence a fundamental understanding of the mitochondrial proteome could


help address this organelle’s role in PE and non-Mendelian inherited

gestational diseases. In the second paper Ishimura et al. (2001) have

prepared a database of 150 plasma membrane proteins using 2-D

electrophoresis of the rat placenta, which are expressed in a stage specific

manner. They have used peptide mapping, amino acid sequence analysis and

mass spectrometry to determine stage specific differentiation modifications

on G protein subunits in the placenta. In the future animal systems will

provide useful proteomics data for the interpretation of human gestational

disease, though it is clear from past human and animal studies that there are

distinct differences not only between the structures of the placenta but in

their endocrinological processes. Some gestational diseases such as PE are

also unique to primates. In the third paper, representing the largest functional

proteomics study by Hoang et al. (2001) the effects of hypoxia on the

cytotrophoblast protein repertoire were examined in first trimester human

cytotrophoblasts against those maintained under standard tissue culture

conditions. It is the cytotrophoblast cells that differentiate and invade the

maternal uterus, whereby forming vascular cells in a process determined by

the increasingly higher levels of oxygen encountered within the uterine wall

(Hoang et al., 2001). The failure of these processes to occur can lead to a

shallow uterine invasion that has been associated with both FGR and PE.

Following 2-D electrophoresis, forty-three spots were identified for MS

which identified differences in the abundance’s of enzymes involved in

glycolysis and those embracing responses to oxidative stress. Glycolysis

enzymes being predicted to rise in response to the increased consumption of

glucose during hypoxia. This study provides new information about the

generalised mechanisms the cells use to respond to changes in oxygen

tension at the maternal foetal interface (Hoang et al., 2001). In the fourth

paper, the proteomics of neurokinin B (NKB) was studied by the comparison

of normal cytotrophoblast cell preparations with those treated with NKB

(Sawicki et al., 2002). Excessive secretion of placental NKB into the

circulation during the third trimester of pregnancy is seen in women with PE

and has been suggested as a cause for this disease (Page et al., 2000). Their

study was performed to determine the possible effects of NKB on normal

placenta, their results indicating that this peptide has multiple actions on the

trophoblast. These actions are consistent with NKB’s role in suppressing

normal antioxidant defences and suppressing proteins that block

proinflammatory responses (Sawicki et al., 2002). These are facets for some

of the placental abnormalities seen in PE.

The development of bioinformatics (the unity of biology and computer

science) should also be mentioned for its importance in providing the

methods for placental proteome profiling. This area has become one of the


fastest growing fields in biological research (reviewed by Luscombe et al.,

2001). The momentum of the Human Genome Project has provided the

opportunity to gather and store vast arrays of valuable data. We have

explored these databases with keywords as simple as ‘placenta’ or sequence

motifs of proteins/peptides to identify partial and uncharacterised cDNA

sequences such as those of expressed sequence tags or Tentative Human

Consensus sequences (Boguski, 1995). 5’ and 3’ rapid amplification of

cDNA ends of candidate placental genes is then performed to reveal the full-

length cDNA sequence of each respective gene which is then translated to

reveal and confirm secretory and functional motifs. By these methods we

have identified several candidate diagnostic markers (Page et al., 2000, Page

et al., 2001). In this manner, bioinformatics can provide a first step

alternative to 2-D electrophoresis in the search for novel proteins/peptides. It

can also provide a convenient method to establish the prediction of protein

variants from encoding donor and acceptor splice sites determined from their

genomic DNA regions. This allows proteins/peptides to be predicted that

may not be yet annotated in the public databases. Subsequently, antibodies

can be raised against such predicted sequences allowing enhanced proteomic

analysis. Bioinformatics has therefore become an integral part of proteomics

being used to predict novel proteins, for the analysis and interpretation of

MS data, the storage, analysis and comparison of gel images and the

prediction of interactions between proteins (Vihinen, 2001).

5. PEPTIDOMICS TO STUDY GESTATIONAL

DISEASE

Peptidomics provides many new challenges beyond the application of

standard proteomics techniques in the characterisation of peptides (Jurgens

& Schrader, 2002). It comes from the recognition that many of the

techniques applied to the study of proteins cannot be employed so easily to

the analysis of peptides. Whereas 2-D electrophoresis in combination with

MS has become the corner stone of most proteomic research there are only a

few approaches to date that describe and tackle the analysis of the

peptidome. Here, are addressed some of the issues we are tackling in the

development of our own peptidomic strategy for the discovery of novel

placental peptide markers.

5.1 Placental peptides

Peptides, oppose to proteins, are typically considered those to be up to

and around 200 amino acid residues in length, that is, those less than 20 kDa


in molecular mass. Examples of different placental peptide sub-groups are

the glycosylated polypeptides such as the non-covalently linked α and βchains of hCG, the single polypeptide chains such as placental lactogen (PL),

the polypeptide cytokines such as TNF-α and the small peptides such as

CRF and NKB. Surprisingly, the placenta has been found to produce many

of the known peptides, which have been found to elicit a wide range of

functions. Typically, these peptides are synthesised as precursors, which

have to be processed by the cell to release the bioactive peptide or peptides

in a highly regulated manner. They generally undergo post-translational

modifications including acetylation, amidation, carboxylation, glycosylation,

phosphorylation and sulphation. Intramolecular disulphide bonding either

along the same chain or to link two or more chains is common. The

precursor has a strongly hydrophobic signal peptide sequence at its NH2

terminus, responsible for translocating it to the endoplasmic reticulum.

Cleavage of this signal sequence in the endoplasmic reticulum forms the

precursor, which is further cleaved, normally at dibasic amino acid residues

(e.g. Arg-Arg, Lys-Arg) to yield the smaller peptide sequences. Prohormone

convertases are responsible for these final cleavages which usually occur in

the Golgi apparatus or in the secretory vesicles. The final secretion of the

peptide occurs either by constitutive secretion where peptides are released

immediately or by regulated secretion where they may be stored prior to

release. Secreted peptides have not only been localised to the foetal side of

the placenta, that is the syncytiotrophoblasts and cytiotrophoblasts of the

villi, but to the membranes of the chorion and amnion, and to the maternal

decidual cells and those of the endothelium. Such peptides are shown to have

diverse effects including the maternal recognition and adaptation to

pregnancy. Whereby, they play an important role in changing the female’s

reproductive system from a cyclic pattern to a pregnancy state, controlling

trophoblast invasion, angiogenesis, growth, metabolism, immune function

and cardiovascular responses.

5.2 Tissue/fluid sampling for gestational disease

Placental peptides secreted into the maternal and foetal circulation,

amniotic fluid and mother’s urine allow their potential detection from a

number of different sites. Consequently, there is the need to establish the

most suitable tissue/fluid to analyse and the best method for their extraction.

Ultimately, non-invasive methods such as maternal venous blood or urine

sampling will be advantageous over many of the existing prenatal screening

techniques. These include amniocentesis, chorionic villus sampling,

cordocentesis and foetal biopsy, which are all very invasive and convey a

significantly higher risk of miscarriage (Scott et al., 2002). However,


definitive diagnosis for chromosomal disorders at present is gained only by

invasive screening as it relies on the culture and karyotyping of cells of

foetal origin. In the genomics/transcriptomics age, the placenta has had to

play the key role for the source material for identifying novel placental

markers (Page et al., 2000). This will continue in the peptidomic age, where

normal placenta can be collected from abortion clinics (legally up to week

24 in the UK) or at term (weeks 37 to 42) following obtaining the

appropriate local ethical approval. Placenta from other time periods can only

be obtained in the UK, if there is a substantial risk to the woman's life or if

there are foetal abnormalities. However, peptidomics extends the repertoire

of source material beyond that of placental tissue, as pathological changes in

a gestational disease may also be detectable in the previously restricted

extracellular fluids of the amniotic fluid, foetal and maternal blood and

urine. However, collection of some extracellular fluids e.g. amniotic fluid

and foetal cord blood is highly invasive. Amniotic fluid is normally only

collected between week 15 to 16 of pregnancy with no more than 15 mls

being taken. While earlier amniocentesis (before 14 weeks) is associated

with significant problems, including increased foetal loss, foetal talipes and a

reduced amniocyte culture rate (Nicolaides et al., 1994). In the case of

cordocentesis for foetal blood, routinely performed after week 18, there is a

significant risk of miscarriage (Tongsong et al., 2000). This makes direct

peptidomic analysis of maternal blood along with maternal urine, the most

convenient and low risk procedure for obtaining an accurate reflection of

secreted placental markers in the maternal body fluids. Such sampling can be

performed much earlier in pregnancy with the hope of reducing the amount

of psychological anxiety and pathological trauma faced by the prospective

parents. Neither could such direct maternal information be inferred

accurately from placental tissue alone as it is estimated that such

measurements would chiefly reflect the higher content of foetal extracellular

fluid/tissue. Lin et al. (1976) estimated that a 400 g placenta would occupy a

312 ml volume, and would contain 144 ml (46%) of foetal blood, of which,

only 36 ml (11.5%) would be derived from the mother.

5.3 Separation and analysis of peptides

In most proteomic studies, peptides are not well portrayed on 2-D

electrophoresis gels and are represented by only a few faint spots with low

staining capacity which can appear less focussed owing to their higher gel

mobility. Moreover, when they are below 10 kDa in molecular mass even on

high percentage polyacrylamide gels small peptides are extremely difficult to

keep within the gel. For this reason liquid chromatography (LC) has been

adopted as the principal separation technique for peptides with reverse phase


and ion exchange being the preferred current adaptations. Samples are

prepared normally by an initial peptide extraction. This is typically

performed by acidifying the sample and conducting a solid phase extraction

(e.g. using Sep-pak cartridges). In the case of plasma and sera samples

additional steps may be taken to deplete the bulk of the abundant sera

proteins such as albumin which can comprise 80% of the sample. Often

these depletion processes include immunoaffinity (Kennedy, 2001), ultra-

filtration (Schulz-Knappe et al., 1997) or gel filtration chromatography

(Schulz-Knappe et al., 2001). Combinations of chromatographic procedures

are used to reduce the complexity of the peptide array, separating either by

hydrophobic interactions or by charge, or both. Such chromatographic

procedures have led to the production of peptide banks containing complete

spectra of fractions from human plasma obtained by ultra-filtration (Schulz-

Knappe et al., 1997). Ultra-filtration from plasma preserves the presence of

naturally occurring peptides, in much the same manner as those collected

from urine that are below the kidney cut-off size. Characterisation of these

peptide banks is performed using MS such as matrix assisted laser

desorption/ionisation time of flight MS (MALDI-TOF-MS) to generate mass

databases based on peptide molecular masses (Richter et al., 1999). Richter

et al., (1999) recorded approximately 5,000 different peptides with 95% of

the detected masses smaller than 15 kDa from one such plasma peptide bank.

Subsequently, MS-MS sequencing, with its much greater resolution power,

revealed the amino acid sequence of many of these circulating peptides. The

combined use of these techniques has been termed peptide trapping (Schulz-

Knappe et al., 2001). Multidimensional chromatography is also applicable to

peptide separation. Such separation involves high pressure LC in the first

dimension, followed by MS in the second dimension resulting in distinct

high resolution peptide mass fingerprints. The MS data of all samples is then

combined into one 2-D diagram, called a peptide display (Schulz-Knappe et

al., 2001). From samples volumes of less than 1 ml, more than 1,000

different peptides have been depicted on each peptide display with regard to

their relative quantities, molecular masses and chromatographic elution

behaviours (Schulz-Knappe et al., 2001).

However, there remain important considerations in the MS analysis of

peptides, as compared to those of proteins. These include the fact that the

number of specific internal proteolytic (tryptic) sites are limiting and hence

generally not available to produce specific peptide fragments. In this regard,

MS analysis of proteins has relied heavily on the DNA sequence databases

produced from both the genomic and the bioinformatic efforts to predict

digested protein fragments. Peptides may be present in many different forms

from their mature precursors, to partially processed precursors, to their fully


cleaved peptides. They may also be present as genetic variants, splice

variants, post-translationally modified or as degradation products. They may

also contain previously unrecognised unique processing sites, that cannot yet

be predicted. Much of this information is not annotated in the current

databases.

6. DEVELOPING DIAGNOSTIC TESTS

The criteria for any placental marker is that it must be 1) accurate with a

good safety profile 2) can be used at the earliest possible period in gestation

allowing for informed choice for pregnancy termination and 3) be rapid so as

not to lead to unnecessary parental anxiety. In its development the following

questions need to be addressed 1) is the marker unique to any one particular

condition and 2) can a consensus be determined from a large longitudinal

study into the merits of a particular marker? There are no studies to guide the

use of placental markers derived from proteomics in the clinic. Clinical

proteomics provides the opportunity to develop and utilise the next

generation of placental diagnostic markers with the prospect of tailoring

some of these towards therapeutic intervention. MS products identified as

candidate diagnostic markers can be the intermediate or end products of

several different processes. These include those of metabolomics (Fiehn,

2002), interactomics (Govorun & Archakov, 2002) and degradomics (Lopez-

Otin & Overall, 2002). Metabolomics comprises the end products of cellular

regulatory processes including those of the processing and the modification

of proteins/peptides. Interactomics relates to the way different proteins and

peptides interact. For example, IGF-I and IGF-II circulate in association with

specific binding proteins (IGF-BPs), and their bio-availability during

pregnancy depends on the proteolysis of their specific IGF-BPs. Proteins can

also be cross-linked such as pregnancy-associated plasma protein-A (PAPP-

A) and the proform of eosinophil major basic protein (Overgaard et al.,

2003). Degradomics applies to the identification of the substrates and

products of protease interaction. Subsequently, there is a constant flux in the

dynamics of protein turnover throughout pregnancy, an important and

missing dimension in current proteomics. In the development of any

diagnostic assay it is meaningful to assess fully the effects of these processes

and whether they may cause interference. Otherwise, such a situation could

lead to poor correlation in the detection assays employed. For instance, the

occurrence of a variety of different molecular/modified forms of the same

protein which are not differentiated in a diagnostic assay, the presence or

absence of a masking binding protein or an association with another protein,

or the presence or absence of circulating proteases could all have profound

effects on the final measurement. Many of these functional proteomic


outcomes/interactions can not be predicted from merely 2-D electrophoresis

or MS. They can only be assessed during assay development and ultimately

from the development of specific antibodies and from the outcomes of large

longitudinal studies. Markers can also be temporary e.g. PAPP-A is a very

good marker for DS in the first trimester (between weeks 10 to 14), but when

measured in the second trimester the results are very similar to those

measured for normal pregnancies (Berry et al., 1997). The diagnostic

window chosen is vital.

Methods based on saturation analysis using antibodies have dominated

diagnostic assay development. The original concepts being based on the

radioimmunoassay. However, these original procedures are impractical in

the modern day clinic, being very time consuming and labour intensive.

Modifications have included the use of two-site immunoassays based on

non-isotopic labels including enzymic, chemiluminescent and fluorescent

labels. The definitive diagnostic test will be one performed using a random

access immunoassay system which can provide a high degree of automation

and speed. The aim being to provide a service (one stop clinic) whereby a

woman can be screened for biochemical markers while she attends her

routine ultrasound scan. Her results being presented at the end of this session

with direct access to immediate advice and counselling. One such

immunoassay system is based on time-resolved-amplified-cryptate-emission

(TRACE) which provides automated, precise and reproducible

measurements within 30 minutes of obtaining a blood sample (Spencer et

al.,. 1999). TRACE is based on a non-radioactive transfer of energy, that

takes place between two fluorescent tracers: a donor, europium cryptate, and

an acceptor, that are each bound to an antibody. This technique has already

been developed for AFP, β-hCG and PAPP-A and for a range of fertility

hormones including follicle stimulating hormone and luteinising hormone.

Proteomics may also see direct application in the clinic, in a similar manner

to those used to obtain serum proteomic patterns for the diagnosis of cancer

(Petricoin & Liotta, 2002). While, microchips of peptides and

peptidomimetic compounds may provide powerful tools in the future for

high-throughput routine laboratory operations (Pellois et al., 2002).

7. CONCLUSIONS

Many of the currently used tests for gestational disease provide only an

estimation of risk as many mothers and foetuses never actually have or will

ever develop the disease. Hence, it is vital to develop new prenatal screening

tests that are more reliable and specific. We believe peptide markers may be

able to fill this niche, however, ideally they should be unique to the


condition and specific to a stage of the disease. It is clear that while peptides

are very promising candidates, there is still much to be learnt. At present, not

all markers are unique to any one particular condition and no consensus has

yet been reached in any study undertaken. The issues are complex and it is

hoped that proteomics/peptidomics will be able to compliment the vast

amount of knowledge already gained from genomic and bioinformatic

studies. Whereby, we will learn fresh data about the way placental markers

are expressed, processed, post-translationally modified, secreted and

metabolised in each of the different gestational diseases. It is anticipated that

this will pave the way forward for the identification of specific targets for the

design of robust, rapid and clinic friendly diagnostic assays. And, finally that

some of these markers may see applications in therapeutic intervention.

ACKNOWLEDGEMENTS

I would like to thank Nicola Bell who has helped in the final proof

reading of this chapter.

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