1881

Electrophoresis 2012, 33, 18811893

1

Manja Wolter
1

Claudia Rower
Cornelia Koy1
Toralf Reimer2
Werner Rath3
Ulrich Pecks3
Michael O. Glocker1
1 Proteome

Center Rostock,
Medical Faculty and Natural
Science Faculty, University of
Rostock, Rostock, Germany
2 Department of Obstetrics and
Gynecology, Medical Faculty,
University of Rostock, Clinic
Suedstadt, Rostock, Germany
3 Department of Obstetrics and
Gynecology, Medical Faculty,
RWTH Aachen University,
Germany

Received January 1, 2012

Revised January 31, 2012
Accepted February 16, 2012

Research Article

A proteome signature for intrauterine

growth restriction derived from
multifactorial analysis of mass
spectrometry-based cord blood serum
profiling
Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does
not reach its genetically given growth potential, resulting in low birth weight. IUGR is
an important cause of perinatal morbidity and mortality, thus contributing substantially
to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the
control group (n = 15) to fractionation by affinity chromatography using a bead system
with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by
MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z
8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively
assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals
in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction
with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of
this proteome signature, apolipoprotein C-III0 , a derivative lacking glycosylation, has been
found more abundant in the IUGR cord blood serum samples, irrespective of gestational
age. Hence, we suggest apolipoprotein C-III0 as potential key-marker of the here proposed
IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density
lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is
discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle
alterations in protein glycosylation need to be considered for improving our understanding
of the pathomechanisms in IUGR.
Keywords:
Affinitymass spectrometry / IUGR / Cord blood analysis / MALDI-TOF MS /
Multiparametric profiling / Proteome signature
DOI 10.1002/elps.201200001

1 Introduction

Correspondence: Dr. Michael O. Glocker, Proteome Center Rostock, University of Rostock, Schillingallee 69, D-18057 Rostock,
Germany
E-mail: michael.glocker@med.uni-rostock.de
Fax: +49-381-494-4932

Abbreviations: AUC, area under curve; CHCA, alphacyano-4-hydroxy cinnamic acid; CTRL, control; DHB, 2,5dihydroxybenzoic acid; HDL, high-density lipoproteins; IUGR,
intrauterine growth restriction; [M+H]+ , protonated molecular ion; MRM, multi-reaction monitoring; m/z, mass to charge
ratio; n-OGP, n-octylglucopyranoside; ROC, receiver operator
characteristics; SGA, small for gestational age; TIC, total ion
count; VLDL, very low-density lipoproteins

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Intrauterine growth restriction (IUGR) affects about 3 to 8%

of all pregnancies. It is defined as a condition in which the
fetus does not reach its genetically given growth potential,
resulting in low birth weight. The pathogenesis of IUGR
still remains unclear. Diminished cytotrophoblast invasion
and uterine spiral artery remodeling leading to placental insufficiency have been hypothesized to play crucial roles in
the development of IUGR. Hence, the pathomechanisms in
IUGR are considered similar to those in preeclampsia by
which the mothers are affected. The latter is a new onset hypertensive disorder in pregnancy with additional proteinuria,
typically occurring after the 20th week of gestation. The assumed similarities in pathogenesis of both diseases might
explain the fact that both conditions occur simultaneously in
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M. Wolter
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up to 30% of the cases [13]. IUGR is clinically diagnosed by a

deceleration of fetal growth velocity in serial antenatal sonographic measurements of fetal biometry (crossing centiles)
[3, 4]. In clinical practice, one has also considered to define
IUGR as a low estimated fetal weight in addition to signs
that reflect placental insufficiency. Of those, compromised
fetal well being (pathological fetal cardiogram or resistance
indices in Doppler sonographic measurements), asymmetry,
and/or reduced amnion fluid index in ultrasonographic examinations have been suggested to be most important [59].
The clinical challenge for obstetricians is to determine the
optimal time point of preterm delivery in order to reach sufficient fetal maturity and preventing fetal death. Hence, IUGR
contributes substantially to medically indicated preterm
birth [3, 5].
Over the last decades, it has become evident that abnormal intrauterine conditions also increase the infants risk for
cardiovascular and metabolic diseases later in life. The association of being born small for gestational age (SGA) and
the development of atherosclerotic cardiovascular diseases
later in life has been shown by several epidemiologic studies
[1012]. Such observations have led to the fetal origins of
disease hypothesis or Barkers hypothesis in that abnormal conditions in the intrauterine environment may cause
long-lasting structural and functional systemic alterations.
But by now, exact mechanisms of fetal programming are
speculative.
Proteome analyses have the potential to identify marker
proteins and/or signatures of proteins of importance for a
given disease state and previous 2D-gel-based analyses of
IUGR cord blood serum samples have shown that inflammatory response proteins, immune response proteins, as well
as proteins related to transport, blood pressure, and coagulation seem to be of importance [13]. In other 2D-gel-based
studies, prominent changes of the sialic acid decoration of
2-HS glycoprotein (also termed fetuin-A) were observed in
IUGR cord blood serum samples when compared to controls
[14] and were discussed as potentially responsible for IUGRassociated complications.
In order to gain deeper insights into protein compositions from cord blood samples which were not within reach
of hitherto performed investigations but might display information by which cord blood serum from IUGR samples could
be characterized and even distinguished from control samples, we have collected cord blood samples from a group of
clinically diagnosed IUGR infants at birth as well as matched
cord blood samples from unaffected newborns. We subjected
all cord blood serum samples to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were then analyzed by MALDI mass spectrometric profiling, as this approach was previously proven successful in multiparametric characterization of blood samples from pregnant women
[1518]. Since it is evident by epidemiological studies that
IUGR neonates are at increased risk for the development of
cardiovascular and metabolic diseases later in life, we secondary aimed to identify differentially abundant ion signals

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2012, 33, 18811893

in order to detect proteins related to atherosclerosis in umbilical cord blood, as identifying those proteins could help
developing a targeted therapy for prevention.

2 Materials and methods

2.1 Patient cohorts and controls
The study was approved by the Ethics Committee of the
RWTH Aachen (EK 119/08). Written informed consent
was obtained from all participating parents. We analyzed
cord blood samples from 30 newborns at the Department
of Obstetrics and Gynecology, University Hospital of the
RWTH Aachen between March 2008 and August 2010. Of
those, 15 healthy infants were born adequate for gestational
age with 11 being born preterm for various reasons, and
15 infants suffered from IUGR with 12 needed mandatory preterm delivery. Gestational age was established on
the basis of the last menstrual period and confirmed by
ultrasonic examination between 10th and 14th week of
gestation. Neonatal birth weight centile was determined
according to the population-based, newborn weight charts
[19]. Sonographic examinations were done antenatally on
R
R
and Voluson 730 Expert
Ultrasound Systems (GE
Logiq 5
Healthcare Systems, Solingen, Germany). The regression
equation including biparietal diameter, femur length, and
head and abdominal circumferences, proposed by Hadlock et
al., was used to estimate fetal weight [20]. IUGR was defined
in accordance to the ACOG guidelines and as described
recently [5, 8]. In addition to an estimated fetal weight below
the 10th percentile, the following criteria had to be fulfilled:
(i) deceleration of fetal growth velocity during the last 4
weeks, (ii) elevated resistance index in umbilical artery
Doppler sonography above 95th percentile or absent or reversed end-diastolic blood flow, (iii) fetal asymmetry (head to
abdominal circumference ratio above the 95th percentile), or
(iv) oligohydramnios (Amniotic Fluid Index < 5 cm). Neonatal weight was assessed postnatally for correct diagnosis of
SGA < 10th percentile. Additional preeclampsia was defined
according to the International Society for the Study of Hypertension in Pregnancy guidelines [21]. The control group
(CTRL) was chosen on the basis to match for gestational age
and gender, and to keep maternal baseline characteristics
similar. Neonatal weight in the CTRL group was appropriate
(within the 10th and 90th percentile). Exclusion criteria were
the following: multiple gestation, fetal anomalies, abnormal
fetal karyotype, patients with clinical or biochemical signs of
infection, positive TORCH screening results, maternal diabetes mellitus/gestational diabetes, or other severe maternal
metabolic disorders, and patients withdrawal from the study.
Important clinical characteristics by which mothers and their
neonates were characterized were maternal blood pressure
and urinary protein content, week of gestation at delivery,
and fetal birth weight centile (Supporting Information
Table S1).
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Electrophoresis 2012, 33, 18811893

2.2 Blood sampling, generation, and storage

of serum aliquots
Blood samples (up to 4.9 mL each) were taken postnatally from the umbilical cord vein using Monovette syringes
R
, Sarstedt, Germany). After in(Serum 4.9 mL Monovette
cubation at room temperature for 1530 min, samples were
subjected to sedimentation of blood cells by centrifugation
at 2000 g at room temperature for 15 min. Serum was aspirated and divided into aliquots (100 L each) which were
stored at 80C. Altogether, time between blood sample collection and storage of serum aliquots averaged below 1 h.

2.3 Protein extract preparation from serum samples

using the profiling kit 100 MB-HIC8
Serum samples were processed according to established protocols [17] using the Profiling Kit 100 MB-HIC8 (Bruker
Daltonik, Bremen, Germany). From each thawed serum sample, 5 L were incubated with 10 L MB-HIC8 binding
buffer and 5 L of MB-HIC8 bead slurry for 1 min. After
washing three times (100 L of wash buffer, each), proteins were eluted with 10 L of elution buffer, consisting
of a 50% ACN solution.

2.4 MALDI-TOF MS profiling of serum proteins and

internal recalibration of mass spectra
After MB-HIC8 extraction, protein solutions (0.5 L each)
were spotted directly onto a stainless steel MTP 384 target
plate (Bruker Daltonik) together with 0.5 L ferulic acid solution (10 mg ferulic acid dissolved in ACN/0.1% aqueous TFA
(33/67, v/v) as matrix. After drying, 0.5 L ferulic acid solution
was added to each sample spot again. Protein samples were
prepared in duplicate for recording first and second measurements (M1/M2) of the first sample work-up and for recording
first and second measurements (M3/M4) of the second sample work-up, respectively. Protein mixtures were analyzed
with a Reflex III MALDI TOF mass spectrometer (Bruker
Daltonik) equipped with the SCOUT source and delayed extraction and operated in linear positive ion mode using an
acceleration voltage of 20 kV [17]. Spectra were recorded in
a mass range from 4 to 20 kDa, respectively, accumulating
900 shots per spectrum. Spectra were externally calibrated
using a commercially available Protein Calibration Standard
(Bruker Daltonik). Furthermore, all mass spectra were internally recalibrated using average masses of ion signals at
m/z 13 762.4 (singly charged and unmodified transthyretin;
uniprot accession number P02766; and at m/z 6631.6 (singly
charged and unmodified apolipoprotein C, uniprot P02654).
Ion signal areas were determined with the ClinProTools 2.2
software (Bruker Daltronik) as described previously [18].

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2.5 Bioinformatic analysis of linear MALDI-TOF MS

ion signals
R
2.2 software (Bruker Daltonik) was
The ClinProTools
used to analyze the spectra from the patients and controls, respectively. Each dataset was generated in duplicate, that is, two independent measurements were recorded
from each sample. The first sample work-up with MBHIC8 beads resulted in datasets M1 and M2, and the second work-up in datasets M3 and M4, respectively. Analysis
for each dataset was carried out independently from each
other. Settings have been specified and retained unchanged
for all measurement analyses for (i) peak picking, which
happened on the total average spectrum of the particular class,
(ii) for signal-to-noise threshold, value was set to 5.00, and (iii)
the value for the relative threshold base peak was set to 0.00.
Smoothing of spectra was performed applying the SavitskyGolay algorithm, width 2.0 m/z for three cycles. Spectra were
recalibrated automatically, taking only those masses into account which occurred in at least 30% of the spectra. The
maximal mass tolerance between reference mass and peak
mass was set to 1500 ppm. Hence, those spectra were excluded from analysis which either could not be recalibrated
due to larger mass differences or which contained too few ion
signals for calibration. Baseline subtraction was performed in
the Top Hat Baseline mode and null spectra exclusion was
enabled. All ion intensities in the spectra were normalized
to their own total ion count (TIC), which was determined
as the sum of all intensities of the spectrum. Subsequently,
all intensities of this spectrum were divided by the obtained
TIC value. Statistical significance was tested with a Wilcoxon
rank-sum test [22] combined with a KruskalWallis test [23],
an AndersonDarling test [24], and an ANOVA test combined
with a Students t-test for two populations [25, 26] on significance levels of 0.05. Graphical representations as box-andwhisker plots [27] were realized using the Origin software
(Version 6.1G, OriginLab Corporation, Northampton, MA,
USA).

2.6 Protein extract preparation from serum samples

by fractionated precipitation
Serum samples were thawed and fractionated precipitation was performed according to described procedures [17].
Briefly, to 10 L of serum sample, 7 L of chilled ethanol
(20C; 40% ethanol, v/v) was added and kept on ice for
10 min. The pellet (fraction I) was collected by centrifugation
with 13 000 g at 4C for 10 min. Proteins dissolved in the
ice-cold supernatant (17 L) were precipitated with addition
of 3 L of chilled ethanol (50% ethanol, v/v). After 10 min
incubation at 20C precipitated proteins (fraction II) were
collected by centrifugation as described above. Proteins dissolved in the ice-cold supernatant (20 L) were again precipitated with addition of 5 L of chilled ethanol (60% ethanol,
v/v). The pellet (fraction III) was collected by centrifugation
as already described. The dissolved proteins (25 L) were
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M. Wolter
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Electrophoresis 2012, 33, 18811893

precipitated with addition of 25 L (80% ethanol, v/v). After

10 min incubation at 20C precipitated proteins (fraction
IV) were collected by centrifugation.
2.7 SDS-PAGE and gel imaging
Proteins from the pellet of fraction IV were redissolved in 20
L of SDS sample buffer (2% SDS, 62.5 mM Tris/HCl (pH
6.8), 10% glycerol, and bromophenol blue) at room temperature. SDS-PAGE was carried out as described [28]. In short,
SDS polyacrylamide gels, 15% T for analyzing proteins in
the low mass range were run for 1 h at 200 V in a MiniPROTEAN II 2D Cell BIO-RAD Electrophoresis System applying the Laemmli continuous buffer system [29]. Gels were
fixed and then stained with colloidal Coomassie Brilliant Blue
G-250 [30]. The broad range marker kit (New England BioLabs, Frankfurt M., Germany) was used as apparent molecular mass calibration. Stained gels were scanned with the
Umax Mirage II Scanner (Umax Data Systems, Willich, Germany) and images were stored as 16 bit tif files. For densitometric image analysis, the software package PhoretixTM
2D Advanced, version 6.01 (Nonlinear Dynamics Ltd,
Newcastle upon Tyne, UK) was used.
2.8 Mass spectrometric peptide mass fingerprinting
Protein bands of interest were excised manually and gel plugs
were subjected to in-gel digestion with trypsin (Promega,
Mannheim, Germany) [16]. Sample preparation of peptide
mixtures was performed on an AnchorChipTM 600/384 target plate [28] using DHB as matrix. Peptide mixtures were
analyzed with a Reflex III MALDI TOF mass spectrometer
(Bruker Daltonik) equipped with the SCOUT source and delayed extraction and operated in positive ion mode using an
acceleration voltage of 20 kV. Spectra were externally calibrated using a commercially available Peptide Calibration
Standard (Bruker Daltonik) as well as internally recalibrated
using the following peptide ion signals derived from trypsin
autoproteolysis: [M+H]+ 842.51, [M+H]+ 1045.54, [M+H]+
2211.10, [M+H]+ 2807.39. Mass spectra were further processed and analyzed with the FlexAnalysis 2.4 and BioTools
3.0 softwares. Database searches were performed against an
in-house SWALL database that consists of Swiss-Prot release
51. and TrEMBL release 34.0 using Mascot version 2.2.07 software (Matrix Science, London, UK) with the following search
parameters: taxonomy: human, peptide tolerance: 60 ppm,
fixed modifications: carbamidomethylation of cysteines, variable modifications: oxidation of methionines, one missed
cleavage site.
2.9 Bioinformatic and biostatistical analysis
of mass spectra

Figure 1. MALDI TOF mass spectrum of a serum sample afR

ter ClinProt
work-up from an IUGR cord blood serum sample.
Mass range m/z 600020 000. The inserts show zooms into the
mass ranges m/z 85009400 and m/z 13 70014 000, respectively.
*
indicates ion signals that were used for internal recalibration;
ferulic acid was used as matrix. For assignments of protein ion
signals, see Supporting Information Table S2.

variance) and expressed as mean and 95% confidence interval

(CI). Differences of serum parameters were tested for significance using MannWhitney test. Association analyses were
performed using Spearmans coefficient of rank correlation
(rho). Sensitivity, specificity, and likelihood ratios were measured on receiver operator characteristic (ROC) plots using
the Origin software (Version 6.1G, OriginLab Corporation)
R
2.2 software.
and ClinProTools
Gel views of mass spectrometric data were generated using an in house visualization tool based on a PHP script
(Linux Command Line Interpreter) [15]. The ion signal intensity threshold was set to 3500. Values below 3500 are encoded
in linear gray scales from 0 (baseline; white) to 255 (maximum; black). Spectra (m/z range 85009050) were imported
as character separated csv files as exported from Flex Analysis
(Bruker Daltonik) after baseline correction.
Hierarchical clustering was performed based on the complete linkage method and Spearmans correlation coefficient
as a measure of similarity. Signal intensities were centered
and scaled row-wise for visualization purposes [31]. Normalized values of selected spots are graphically represented in
heat maps.
In order to evaluate the minimal required sample size
that is needed to discriminate the group with IUGR from the
control group on the basis of the obtained data, we carried
out a power analysis [32] using the GPower 3.0.10 statistical
software. We chose a type I error () of 0.05 and a type II
error () of 0.20 in a comparison of two means which is
typically suggested for this type of analysis. Thus, we achieved
a minimal required sample size with a power (1-) of 80%
and a level of significance below 0.05.
2.10 Mass spectrometric peptide sequencing

Data analysis was carried out using the statistical packages

SAS Version 9.1 Software (SAS Institute, Cary, NC, USA).
Clinical data were evaluated by two-way ANOVA (analysis of

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Peptide mixtures from in-gel digests (1 L) were mixed

with matrix solution (1 L) on an AnchorChipTM 600/384
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Figure 3. Distribution of quotient values for selected ion intensity

R
differences from measurements 1 and 2 after ClinProt
work-up
of cord blood serum samples depicted as box and whisker plots.
(A) m/z 8205 and m/z 8766. (B) m/z 8766 and m/z 13 945. (C) m/z
8766 and m/z 9422. (D) m/z 16 001 and m/z 13 883. (E) m/z 15
308 and m/z 13 883. (F) m/z 15 129 and m/z 13 883. The boxes
represent the 25th75th percentiles. The horizontal lines within
the boxes represent the median, the small squares indicate the
mean. The whiskers specify the 5th and 95th percentiles, and the
crosses indicate the 1st and 99th percentiles. Dashed lines mark
selected cut-off values. CTRL, control.

Figure 2. Virtual gel view of MALDI mass spectra in gray scale

mode. Mass range m/z 85009000. Spectra were recorded in duplicate from healthy control cord blood serum samples (rows 1
15) and from IUGR samples (rows 1630). After background subtraction and normalization, the baseline is represented in white
color (intensity 0) and saturated ion signals in black color (intensity 1). The region around the ion signal with m/z 8766 is boxed.
Patient numbering as in Supporting Information Table S1.

target plate. The matrix solution contained 5 mg/mL 2,5dihydroxybenzoic acid (DHB) which were dissolved in 1 mL
of solvent that consisted of 50% ACN and 50% of a 0.1%
TFA solution (v/v). MS/MS spectra were recorded on an Axima MALDI QIT ToF mass spectrometer (Shimadzu Biotech,
Manchester, UK) utilizing a nitrogen pulsed laser (337 nm,
35 ns pulse length) and employing a three-dimensional
ion trap supplied by helium (pulsed flow gas) for collisional cooling and argon (collisional gas) for collisionally
induced dissociation (CID) [33, 34]. Precursor ions were excited with off-resonance sinusoidal waveforms whereas at
the same time argon was allowed to enter the trap. For
fragmentation, the width of the precursor ion selection window was dependent on the mass of the precursor ion and

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

varied between 2 and 10 Da. Spectra were externally calibrated with a manually mixed peptide standard consisting
of bradykinin (17), [M+H]+ 757.39; angiotensin II, [M+H]+
1046.53; angiotensin I, [M+H]+ 1296.68; bombesin, [M+H]+
1619.81; N-acetyl renin substrate, [M+H]+ 1800.93; ACTH
(117), [M+H]+ 2093.08; ACTH (1839), [M+H]+ 2465.19;
somatostatin, [M+H]+ 3147.46; insulin (oxidized beta chain),
[M+H]+ 3494.64; as well as internally recalibrated using
known ion signals of the MS/MS spectrum. Further processing and analysis of the MS/MS spectra was performed with
the LaunchpadTM software, version 2.8.4 (Shimadzu Biotech).

2.11 ELISA assay for apolipoprotein C-III

A commercially available ELISA test kit (AssayMax Human
ApoC-III ELISA Kit, St. Charles, MO, USA) was used in order to quantitatively analyze ApoC-III expression in sera. All
measurements were performed in duplicate. The absorbance
was measured at 450 nm, using the Rosys Anthos 2010 instrument (Anthos Labtec Instruments, Austria).
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M. Wolter
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Figure 4. Hierarchical clustering analysis with quotient values derived from protein ion signal intensities. Duplicate measurements
from control samples are clearly sorted to the CTRL group (left)
and IUGR samples to the IUGR group (right), except for control
samples 14 (one sample) and 15 (both samples) that are allocated
to the IUGR group, and IUGR samples 16 (both samples), 17 (one
sample), and 18 (one sample) that are grouped to the controls.
Quotients AF from top to bottom as in Fig. 3 (indicated at the
right). Patient numbering as in Supporting Information Table S1.

3 Results
3.1 Clinical characteristics of patients and donors
Clinical parameters by which the study group (IUGR) and the
control group (CTRL) could be characterized include both, information about the fetus/infant and the mothers. Important
facts on the mothers are maternal age (IUGR: 30.4 years
(95% CI 26.934.0), CTRL: 31.7 years (95% CI 28.333.9)),
maternal BMI (IUGR: 24.2 (95% CI 22.426.1), CTRL: 23.7
(95% CI 20.826.6)), primipariety (IUGR: 80%, CTRL 27%),
and smoking status of the mother (currently smoking IUGR:
27%, CTRL 20%). The mean week of gestation at delivery
was 33.0 wk (95% CI 30.535.5) for the IUGR group, and
32.8 wk (95% CI 30.235.3) for the CTRL group (Supporting
Information Table S1). Mean fetal weight was 1383 g (95%
CI 10051761) in IUGR and 2129 g (95% CI 15842673)
for CTRL. By definition, IUGR fetuses were born with birth
weight below the 10th percentile (IUGR: mean 4.0 (95% CI
2.55.5), CTRL: 50.7 (95% CI 40.860.5)).
Blood samples were taken postnatally from the umbilical
cord vein and serum was prepared and divided into aliquots
according to standardized protocols. Altogether, time between blood sample collection and storage of serum aliquots
averaged below 1 h.

Electrophoresis 2012, 33, 18811893

using volatile aqueous buffer systems used for preparation of

the samples on MALDI-MS targets.
MALDI-MS measurements were recorded in duplicate,
that is, from one preparation two spectra were collected for
each sample, generating a set of 60 spectra (series MS1 and
MS2). A typical mass spectrum showed more than 60 ion
signals that were reproducibly recorded in the mass range
between m/z 4000 and m/z 25 000 (Fig. 1), forming two
groups of intense ion signals. On average signal-to-noise ratios of 10:1 and better were recorded for small ion signals
(e.g. the lowest S/N in the IUGR group of 6:1 was observed
at m/z 8766 and at m/z 13 945, respectively).
The first group of ion signals was observed in the mass
range m/z 6000 and m/z 10 000 correlating to predominantly
doubly charged (protonated) proteins, such as transthyretin
and its modifications (Supporting Information Table S2). But
in this mass range also singly charged ion signals of small
proteins (e.g. apolipoprotein C-III) were observed.
The second group of ion signals was present in the mass
range of m/z 13 000 to m/z 18 000 containing predominantly
singly charged ion signals. Hemoglobins and apolipoprotein
A-II are prominent examples of the proteins that were causing
the observed ion signals (Supporting Information Table S2).
Protein assignments of ion signals were made possible by
comparison with literature references [17, 3540].
As all spectra showed high similarities to each other,
semi-quantitative differential analysis of ion signal intensities
was found feasible using a statistical approach. According to
the calculated p-values, the best differentiating singly charged
ion signals between the two sample groups were found at m/z
8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z
16 001 (Table 1).
From these ion signals as well as from two ion signals
at m/z 9422 and m/z 13 883 which were found reproducibly
present in all spectra (so-called landmark signals), the areas
under the ion signals were determined (Supporting Information Table S3). In order to reproduce the results, a second
preparation was conducted, leading to a second set of measurements (series MS3 and MS4; Supporting Information
Table S4). This second set of spectra was later used to apply
the discrimination rules that were determined using the first
set of data (see below).

3.3 Test accuracy estimation upon multiparametric

analysis
3.2 Serum profiling by MALDI TOF MS analysis and
patient screening
All cord blood serum samples were individually subjected to
protein fractionation using hydrophobic bead surfaces. This
procedure proved very efficient to remove highly abundant
serum protein species, such as albumin and immunoglobulins that together make up more than 70% of the serum
protein content. The resulting mixture was reflecting serum
proteins (i) of moderate abundance, (ii) with strong binding
affinities to the bead surface, and (iii) which could be eluted

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Because of the close similarities of the spectra, that is, number

of ion signals and relative intensities of them, independent
of their clinical grouping into either the CTRL group or the
IUGR group, ion signal areas that were differentiating between the two groups were also of low intensity in all spectra
(Fig. 2). By using a gel view image, even small but clearly
present differences in intensities (areas) of small ion signals
can be visualized in the vicinity of large (nondifferentiating)
ion signals by distinct gray values that represent the respective intensities (area values). The ion signal at m/z 8766, for
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1887

Table 1. Statistical data of ion signals selected for multiparametric analysis

Entrya)

1
2
3
4
5
6
7
8

m/z

8205
8766
13945
15129
15308
16001
9422
13883

PTTAb)

0.0000155
0.000138
0.000138
0.0000331
0.0000155
0.00321
0.00105
0.00364

PWKWb)

<0.000001
0.0000188
0.0000775
<0.000001
<0.000001
0.00000437
0.00122
0.000191

PADb)

0.00000845
0.534
0.95
<0.000001
<0.000001
<0.000001
0.18
0.183

DAvec)

201.55
28.06
29.13
830.61
233.72
611.11
244.12
33.6

Peak area [a.u.]/standard deviation

CTRL

IUGR

368.66/168.80
48.58/14.17
74.94/20.14
597.34/264.16
165.12/64.22
394.64/242.18
721.47/249.24
98.64/29.63

167.11/61.73
76.64/28.70
45.81/27.34
1427.95/759.19
398.84/199.50
1005.74/895.92
477.34/237.46
65.05/44.79

a) Entries 16: best differentiating ion signals according to peak statistics; entries 7, 8: most robust ion signals according to peak areas.
b) PTTA, p value from combined paired Students t-test and ANOVA test; PWKW, p value from combined Wilcoxon rank-sum test and
KruskalWallis test; PAD, p value from AndersonDarling test.
c) DAve, difference between the maximal and the minimal average peak area/intensity of all classes.

Figure 5. SDS-PAGE analysis of IUGR cord blood serum proteins.

Protein bands (colloidal Coomassie blue staining) after fractionated precipitation (lane 2) are shown. Mass spectrometrically
identified proteins in the bands (numbered 111 at the right) are
given in Table 4. The locations of marker proteins with known
apparent masses are given at the left (lane 1).

example, was consistently found more intense in the serum

samples from the IUGR group with respect to the serum
samples of the controls.
However, the area value distributions of just one ion
signal were not discriminating enough to convincingly sort


C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

a given spectrum into either the IUGR or the CTRL group.

By contrast, it turned out that the two most robust singly
charged ion signals and the six best differentiating singly
charged ion signals carried enough information for accurate
sorting of individual spectra, that is, of individual samples,
after applying rules by which the relations of the respective
ion signal intensities were considered.
The simplest rules that we applied were to relate the
selected ion signals pairwise, forming quotients of the areas
of the respective ion signals at (A) m/z 8205 and m/z 8766,
(B) m/z 8766 and m/z 13 945, (C) m/z 8766 and m/z 8422, (D)
m/z 16 001 and m/z 13 883, (E) m/z 15 308 and m/z 13 883,
as well as (F) m/z 15 129 and m/z 13 883. The resulting
distributions of the ratios were determined for each of the
six pairs (Fig. 3) and showed in fact significant differences
(p-values below 0.01) in all six cases.
Next, cut-off values (dashed lines in Fig. 3) were determined for all six signal intensity ratios such that they were
located between the upper 75% quartile of the group where
lower values were obtained (CTRL group) and below the 25%
quartile of the other group (IUGR) where on average higher
values were calculated for the respective ratios of ion signal
areas. Then, it was tested whether in a given sample spectrum the respective cut-off value was reached or not. When
the value of one quotient was higher than the respective cut
off, a score of 1 was given to this respective sample. In the
contrary case, the score for this sample was set to 0. This
check was carried out for each of the six ion signal ratios
independently. Hence, each spectrum, that is, each sample,
could ultimately reach a cumulative score between 0 and
6.
Finally, it was determined that a cumulative score above
2 was sorting the respective spectrum (sample) into the
IUGR group. Applying these grouping rules for the values of the first measurement series (MS1 and MS2) enabled a clear-cut separation of IUGR samples into the
IUGR group and all control samples into the CTRL group
(Table 2).

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Electrophoresis 2012, 33, 18811893

Table 2. Statistical significance of mass spectrometric profiling results

Measurement True
False
True
False
Sensitivity Specificity False
False
Positive
Negative
ROC
positive positive negative negative
positive rate negative rate predictive value predictive value AUC c)
MS 1+2a)
MS 3+4b)

28
26

1
2

29
28

2
4

0.93
0.87

0.97
0.93

0.03
0.07

0.07
0.13

0.97
0.93

0.94
0.88

0.99
0.91

a) Values from the first measurement series (MS1 and MS2) of the first sample work-up.
b) Values from the second measurement series (MS3 and MS4) of the second sample work-up.
c) ROC, receiver operator characteristics; AUC, area under curve.

both were controls according to clinical data (cf. Supporting Information Table S1). Interestingly, the IUGR group
seemed much more homogenous except for samples 29 and
30 (at the very right in Fig. 4). These samples obviously contained too much hemoglobin as judged by their reddish color,
presumably because of artifactual disruption of red blood cells
during serum preparation. Therefore, these two samples were
excluded from subsequent subgroup analyses. As indicated,
the control group seemed more heterogeneous, consisting
of three major groups. Higher heterogeneities in the control
groups were also observed in earlier studies [17] and is attributed to the fact that in general clinical inclusion criteria
are well defined for the disease under study but less well
defined for controls (absence of disease).
Figure 6. MALDI-QIT-TOF MSn sequencing result after fragmentation of peptide ion signal at m/z 1196.61 Determined partial
amino acid sequences are depicted and were assigned to the
apolipoprotein C-III-derived peptide comprising amino acids 41
51. Amino acid residues are depicted in single letter code. The
mass spectrometric fragment ions from the B-type ion series, the
Y ion series, as well as typical losses of water (* ) are indicated.
#, not identified. DHB was used as matrix.

Ultimately, the scoring system resulted in a sensitivity of

0.93, a specificity of 0.97, a false positive rate of 0.03, a false
negative rate of 0.07, a positive predictive value of 0.97, and a
negative predictive value of 0.94 for the measurement set used
for determining the separation rules (retrospective analysis).
Similar results were obtained for the test set (measurement
series MS3 and MS4; prospective analysis) applying the exact same rules for spectra classification but using the newly
determined area values. With both preparations, the AUC
values of the ROC characteristics of either measurement series were better than 0.9, which is considered satisfactory for
clinical purposes.
All six quotient value distributions AF were also used
for hierarchical clustering of the individual samples from the
first preparation (Fig. 4) which resulted in clear separation of
the samples according to their clinical classification.
Samples located at the left of the hierarchical tree were
belonging to the controls, except for the two measurements
from samples 16. In addition, only one measurement from
sample 17 and one from sample 18 was assigned to the control group although clinically assigned as being IUGR samples. Accordingly, samples located to the right were assigned
as IUGR samples with the exception of both measurements
from sample 15 and one measurement from sample 14 that

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.4 Subgroup analysis of ion signal intensity

differences with respect to gestational age
When analyzing cord blood protein compositions, one has
to consider that patients suffering from severe IUGR are often delivered preterm. Thus, the careful selection of a control
group matched for gestational age at delivery is required to
avoid confounding, because prematurity per se is a pathologic
condition and serum parameters might be biased by factors
leading to premature labor. We therefore tested whether the
protein ion signals that were included into our marker signature for IUGR were affected by gestational age and split
patient samples (cf. Supporting Information Table S1) into
two groups, that is, samples that were obtained from individuals with less than 34 weeks of gestation were separated
from those after 34 weeks of gestation. Next, the mean values of intensities from our signature ion signals and also
the differences of the means were compared to each other
(Table 3).
Intensity differences of the ion signals at m/z 8205, m/z
13 883, m/z 13 945, m/z 15 129, and m/z 15 308 increased
with gestational age. The one ion signal whose differences
in intensities between the IUGR group and the controls diminished in the late samples (i.e. more than 34 weeks of
gestation) as compared to the early stages was that with m/z
16 001. Interestingly, the only ion signal whose intensity difference was not significantly affected by gestational age was
that at m/z 8766. Hence, the protein that originated this ion
signal was considered as being of further interest.
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Electrophoresis 2012, 33, 18811893

1889

Table 3. Subgroup analysis of differentiating ion signal intensities from cord blood samples with respect to gestational age

Ion signal [m/z]

8205
8766
13 883
13 945
15 129
15 308
16 001

Gestation < 34 weeksa)

Gestation > 34 weeksa)

Differencesb)

CTRL

IUGR

CTRL

IUGR

<34 weeks

>34 weeks

311.68
48.18
93.49
70.20
668.38
182.45
470.12

161.62
77.82
74.28
54.04
1204.74
350.01
707.27

454.14
49.16
106.38
82.05
490.78
139.11
281.41

175.36
74.87
51.19
47.65
1326.81
369.48
397.74

+150.06
29.64
+19.21
+16.16
536.36
167.56
237.15

+278.78
25.71
+55.19
+34.40
836.03
230.37
116.33

a) Mean values of ion signal intensities from measurement series MS1 and MS2 (cf. Supporting Information Table 2).
b) CTRL value minus IUGR value.
Table 4. Identified proteins in SDS-PAGE analysis after fractionated precipitation of serum proteins from cord blood

Band
numbera)

Accession
numberb)

Protein name

Scorec)

RMS
[ppm]d)

Matchede)

Coverage
[%]f)

Molecular
mass [Da]g)

1
2
3
4
5

P02765
P06727
P02766
P02647
P69891
P69892
O95445
P02753
P02652
P02766
P69891
P69892
P02656

Alpha-2-HS-glycoprotein
Apolipoprotein A-IV
Transthyretin
Apolipoprotein A-I
Hemoglobin subunit gamma-1
Hemoglobin subunit gamma-2
Apolipoprotein M
Retinol-binding protein 4
Apolipoprotein A-II
Transthyretin
Hemoglobin subunit gamma-1
Hemoglobin subunit gamma-2
Apolipoprotein C-III h)

93
377
92
246
172
172
158
127
100
197
158
158
48

37
26
53
17
29
29
34
23
24
34
19
19
25

8
30
5
23
11
11
11
11
6
9
10
10
4

22
62
55
64
74
74
35
68
64
69
74
74
51

40 098
45 371
15 991
30 759
16 187
16 173
21 582
23 337
11 282
15 991
16 187
16 173
10 845

6
7
8
9
10
11

a) Band numbers refer to gel in Fig. 5.

b) Uniprot data base entry.
c) Probability based MOWSE score.
d) Root mean square error.
e) Number of ion signals that match with database entry.
f) Sequence coverage of ion signals with respect to database entry.
g) Calculated from database entry.
h) Confirmed by MALDI-QIT-TOF-MS/MS sequence analysis (cf. Fig. 6).

3.5 Evaluating apolipoprotein C-III results by

SDS-PAGE and ELISA
In order to substantiate protein assignments of the serum
samples, we carried out an SDS-PAGE analysis after fractionated precipitation of serum proteins (Fig. 5). Fractionated
precipitation eases SDS-PAGE analysis of low abundant proteins but because of difficulty to reproduce precipitation conditions is not considered to be precise enough to be applicable
for differential quantitative analysis. In addition, separation
power of SDS-PAGE is not sufficient to guarantee avoidance
of comigration of serum proteins. About 11 strongly stained
bands were visible in the gel with the protein mixture in the
mass range between 55 kDa and 6 kDa in both, the IUGR
samples and in the control samples (lane 2 shows the protein
distribution of a representative patient sample). Proteins in
these intense bands were subjected to mass spectrometric
identifications after in-gel digestion with trypsin.

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

We found that band 11, migrating at 9 kDa apparent

mass, contained apolipoprotein C-III (P02656) as the major compound (Table 4); although with low identification
score. Band 4 was determined to contain apolipoprotein AI (P02647). Band 2 contained apolipoprotein A-IV (P06727),
and band 1 at ca. 53 kDa apparent mass contained alpha2-HS-glycoprotein (P02765; fetuin A). With the exception
of apolipoprotein C-III in band 11, all other protein identifications were obtained with good scores (above 90), indicating that the identified proteins were in fact the major
compounds in their respective bands. However, differentiating between hemoglobin subunit gamma 1 and hemoglobin
subunit gamma 2 in bands 5 and 10, respectively, was not
possible.
In order to confirm the identification result for
apolipoprotein C-III from band 11, we performed mass spectrometric peptide sequencing of the ion with m/z 1196.61
using a MALDI-QIT-TOF MSn instrument. The MS/MS
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1890

M. Wolter
et al.

spectrum (Fig. 6) showed a dominating ion signal at m/z

638.37 which can be explained by cleavage of a D-G bond
(Y 6 fragment) that is present in the suspected amino acid
sequence comprising amino acids 4151 (sequence: GWVTDGFSSLK). Due to unique instrument specificities, further
sequence-specific fragment ion signals, however with much
lower intensities, were generated in the QIT ion trap together
with internal fragments and loss of water ion signals, confirming the sequence of peptide 4151 from apolipoprotein
C-III.
As the ion with m/z 1196.61 is reported to belong to
a proteotypic peptide used for MRM assays [41], the identification of apolipoprotein C-III was considered solid. As no
additional proteins could be identified in this band, it seemed
likely that the major protein component in this band was in
fact apolipoprotein C-III.
Since the SDS-PAGE-derived protein identification results were in agreement with the MALDI-MS-based assignments, and since the ion signal intensity differences of the
signal at m/z 8766, assigned to apolipoprotein C-III0 were
distinct but not affected by gestational age, we initiated
quantification experiments for apolipoprotein C-III by ELISA
using a commercially available assay (Table 5).
In ELISA analyses, the apolipoprotein C-III concentrations in cord blood samples varied approximately by a factor
of seven between individuals, ranging from 10.1 g/mL to
69.5 g/mL. Although there seemed to be a trend for decreased apolipoprotein C-III levels in the IUGR group, distinctly different apolipoprotein C-III concentrations between
IUGR samples and controls were not evident (p > 0.05).
Obviously, the mass spectrometrically observed differences between the ion signal intensities at m/z 8766 which
were attributed to belonging to apolipoprotein C-III0 , were
not present in ELISA analyses. This lack of correlation may be
explained by the fact that this ELISA assay does not differentiate between apolipoprotein C-III sialylation forms. Hence,
we estimated the relative abundances of all four apolipoprotein C-III-derived ion signals (m/z 8766 (Apo C-III0 ), m/z
9134 (Apo C-III0  ), m/z 9422 (Apo C-III1 ), and m/z 9713 (Apo
C-III2 ); Table 5), by summing up their ion signal areas to
100%. In this apolipoprotein C-III-focused investigation we
again found that the best differentiating protein species between samples from the IUGR group and the controls was
apolipoprotein C-III0 . Whereas in the CTRL group, the relative amount of apolipoprotein C-III0 was around 34% (with
exception of sample 15) of the total apolipoprotein C-III, the
relative amount of apolipoprotein C-III0 in the IUGR group
was around 810%.

4 Discussion
We recently reported on affinity-based, multiparametric
MALDI-TOF MS analysis of blood serum samples as an overall robust and valid method for both, biomarker discovery,
and for diagnostic purposes in pregnancy-related disorders
[17, 18]. We now adapted this method for the analysis of um
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2012, 33, 18811893

bilical cord blood samples by focusing on neonates that were

born after pregnancies were complicated by IUGR.
To our knowledge, two studies using proteomic approaches for the analysis of umbilical cord blood comparing
IUGR and control groups have been reported [13,14]. In both,
differential 2D gel electrophoresis had been performed followed by peptide mass fingerprinting to identify the proteins
of interest. In the first study, 18 protein spots were reported
to be differentially expressed in umbilical cord serum samples as compared to controls. The identified proteins from
these spots were discussed as being involved in the coagulation process, immune mechanisms, blood pressure regulation, and iron or copper homeostasis [13]. Unfortunately, the
authors did not provide sufficient information about neither
additional antenatal sonographic examinations to confirm the
diagnosis IUGR nor to the week at delivery. According to our
experience, IUGR fetuses are born significantly earlier than
normal and, if compared to a full-term control group, detected
protein alterations in cord blood serum might be overlayed
by protein composition changes that are due to the gestational age of the fetus at delivery. The other study focused
on late-onset IUGR where infants were born round about
after 39 weeks of gestation. Here, 16 proteins were reported
to be differentially expressed. The most interesting finding
of this study was that the negative acute phase parameter 2 HS glycoprotein (fetuin-A) was decreased in IUGR samples.
Interestingly, the authors found that in the IUGR samples
fetuin-A forms, lacking O-linked sialic acid residues, were
uniquely present while absent in plasma samples of the control group [14].
In contrast to these two published studies, we here focused on cord blood serum proteins of low mass range which
usually escape detection by 2D gel electrophoresis but can be
interrogated by affinity-based MALDI-TOF MS as a screening tool. Our results show that protein abundance differences
between IUGR cord blood samples and CTRL samples can
be reliably determined using an affinity-enrichment procedure combined with MALDI-MS profiling. Even more, mass
spectrometry-based multiparametric analysis of cord blood
samples is capable of differentiating individual samples from
the IUGR group from those of the control group with high
confidence.
We recently hypothesized that alterations in cord blood
composition toward an atherogenic phenotype secondary to
placental insufficiency may underlay at least in part the predisposition of the development of cardiovascular diseases in
IUGR [42] as we found atherogenic lipoprotein indices and
total triglyceride concentrations to be significantly increased
in IUGR as compared to controls. In this context it is of interest that we found in this study that apolipoprotein C-III0 was
the most prominent apolipoprotein C-III derivative with differential abundance. Apolipoprotein C-III is a 79 amino acid
constituent of both apo B- and apo A-I-containing lipoproteins [43, 44]. In normolipidemic individuals apolipoprotein
C-III is found in HDL and VLDL particles whereas in patients with elevated plasma triglycerides it is associated predominantly with triglyceride rich lipoproteins associated with
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1891

Table 5. Total apolipoprotein C-III concentrations and relative amounts of sialylated species in cord blood samples

Patient

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Apo C-III
[g/mL]a)

54.68
69.49
33.21
16.96
21.94
43.33
26.55
15.46
22.04
25.05
22.44
27.43
18.79
35.98
15.17
15.94
26.01
10.11
25.93
12.99
24.74
18.98
46.64
14.96
26.87
10.32
34.43
20.93
34.11
15.12

Standard
deviation

0.060
0.018
0.042
0.028
0.004
0.032
0.043
0.013
0.006
0.015
0.016
0.014
0.016
0.025
0.023
0.005
0.029
0.000
0.003
0.011
0.018
0.004
0.016
0.008
0.015
0.001
0.021
0.018
0.045
0.006

Apo C-III0
[%]b)

Apo C-III0
[%]b)

Apo C-III1
[%]b)

Apo C-III2
[%]b)

MS 1

MS 2

MS 1

MS 2

MS 1

MS 2

MS 1

MS 2

4
4
4
5
2
2
3
5
2
5
7
6
4
2
8
9
7
9
5
9
9
8
7
10
11
11
6
9
10
21

3
3
3
4
3
2
4
8
2
5
8
5
4
2
9
8
9
8
7
8
9
7
6
9
15
10
5
11
9
18

5
5
5
5
6
6
8
8
10
7
8
5
6
4
6
8
6
4
6
4
3
5
6
5
5
10
4
5
11
7

5
6
5
5
5
7
7
7
10
8
8
5
6
4
8
7
5
5
7
4
4
5
6
5
6
11
4
6
11
9

49
56
60
64
55
60
56
58
57
58
53
56
58
57
57
49
58
43
60
50
44
46
65
48
55
55
50
50
67
43

49
57
60
61
54
59
56
58
57
55
56
59
56
54
56
50
56
40
58
49
44
46
63
47
49
53
50
50
68
49

42
35
31
26
37
32
33
29
31
30
32
33
32
37
29
34
29
44
29
37
44
41
22
37
29
24
40
36
12
29

43
34
32
30
38
32
33
27
31
32
28
31
34
40
27
35
30
47
28
39
43
42
25
39
30
26
41
33
12
24

a) ELISA determinations.
b) Relative peak areas of ion signals at m/z 8766 (Apo C-III0 ), m/z 9134 (Apo C-III0  ), m/z 9422 (Apo C-III1 ), and m/z 9713 (Apo C-III2 ),
respectively.

LDL and VLDL particles [45]. Under physiological conditions,

apolipoprotein C-III exists in four major species designated
as C-III0 , C-III0  , C-III1 , and C-III2 , where the subscript indicates the number of sialic acid residues. In previous studies,
it was found that apolipoprotein C-III2 binding to VLDL was
2-fold greater as compared with C-III0 and C-III1 binding,
and simultaneously displayed a decreased inhibitory effect
on the lipolysis stimulated receptor (LSR) that is involved in
the clearance of triglyceride-rich proteins [46]. From these
observations and from animal experiments apolipoprotein
C-III0 has been suggested to delay triglyceride catabolism
linking it to hypertriglyceridemia and to atherosclerotic diseases [47, 48]. Moreover, it was reported that loss of sialic
acid residues from lipoprotein particles in general can induce cholesterol-ester accumulation in human aortic smooth
muscle cells. Thus, desialylation of lipoproteins was considered directly involved in the early stages of atherogenesis characterized by foam cell formation [49]. The observation of desialylated apolipoprotein C-III0 being increased in

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

IUGR samples stands in agreement to these observations

and might provide at least a partial explanation for the increased triglyceride levels in cord blood serum. Hence, this
assumed association between apolipoprotein C-III0 protein
abundance, and triglyceride levels in cord blood samples is
worth to be analyzed further and is current focus of ongoing
studies.
Even more, since another protein, fetuin-A, was present
in its desialylated form in IUGR cord blood samples as well
[14], it is tempting to speculate whether enzymatic desialylation and/or release of immaturely sialylated protein derivatives into circulation might be regarded as a common phenomenon in the pathogenesis of IUGR. Protein-bound sialic
acid residues are involved in a variety of physiological and
pathological processes including modulation of fertilization
and development, alteration of immune response [50], oncologic diseases, and brain development (for reviews see
[51, 52]). Of particular interest is the finding that genetic
elimination of sialic acid production in the mouse results in

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M. Wolter
et al.

embryonic lethality [53]. Thus, studying alterations in sialic

acid metabolism seems likely to provide a novel approach
for improving our understanding of the pathomechanisms
in IUGR.
This work was supported by grants from the University of
Rostock and the University of Aachen. The authors like to express their thanks to Mrs M. Ru, Mr. M. Kreutzer, and Mr. F.
Steinbeck for valuable methodological help.
The authors have declared no conflict of interest.

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