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Effect of sialic acid content on glycoprotein pI analyzed by two-dimensional

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DOI: 10.1002/elps.200900764 · Source: PubMed

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Electrophoresis 2010, 31, 2903–2912 2903

Sı́lvia Barrabés1 Research Article

Ariadna Sarrats1
Esther Fort2
Rafael De Llorens1 Effect of sialic acid content on glycoprotein
Pauline M. Rudd3
Rosa Peracaula1
pI analyzed by two-dimensional
1
Unitat de Bioquı́mica i Biologia
electrophoresis
Molecular, Departament de
Biologia, Universitat de Girona, 2-DE is broadly used for quantitative analysis of differential protein expression in complex
Girona, Spain mixtures such as serum samples or cell lysates. PTMs directly influence the 2-DE pattern,
2
Unitat de Digestiu, Hospital and knowledge of the rules of protein separation is required in order to understand the
Universitari Dr. Josep Trueta,
Girona, Spain protein distribution in a 2-DE gel. Glycosylation is the most common PTM and can modify
3
Dublin-Oxford Glycobiology both the molecular weight and the pI of a protein. In particular, the effect of charged
Laboratory, National Institute for monosaccharides (mainly sialic acids, SAs) on the 2-DE pattern of a protein is of major
Bioprocessing, Research and
interest since changes in sialylation are regularly observed in comparative studies. Little is
Training, Conway Institute,
University College Dublin, known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this
Dublin, Ireland shift is the same for all glycoproteins. To address this issue, this study examined the
influence of SA on the 2-DE pattern of three serum glycoproteins (haptoglobin, a1-anti-
trypsin and ribonuclease 1), which N-glycan chains had been previously characterised, and
Received December 21, 2009
Revised May 27, 2010 reviewed existing bibliographic data. The SA content of the different glycoforms of a
Accepted June 8, 2010 glycoprotein showed a negative linear correlation with the pI, although the slope varied
among the studied glycoproteins. We also described a positive correlation between the
protein pI and the pI decrease per SA molecule.

Keywords:
2-DE / Glycoprotein / Sialic acid DOI 10.1002/elps.200900764

1 Introduction acids (SAs) or sulphated monosaccharides, are added to N or

2-DE is an important tool for comparative studies of protein
expression. Many different kinds of PTMs can alter the 2-DE
pattern of a given protein [1]. PTMs such as phosphorylation
[2, 3], deamidation [4] or glycosylation [5] can modify the
molecular weight of a protein, its pI or both parameters. The
combination of all these factors leads to great complexity in
the migration pattern of proteins in the 2-DE map and
increases the difficulty of its interpretation.
It is estimated that 50% of all proteins are glycosylated,
and among the currently known PTMs, glycosylation has
been widely recognised as one of the most influential
parameters in the alteration of protein 2-DE patterns [6].
This modification alters the apparent molecular weight of a
protein, and when charged monosaccharides, such as sialic
Correspondence: Dr. Rosa Peracaula, Unitat de Bioquı́mica i
Biologia Molecular, Departament de Biologia, Universitat de
Girona, Campus de Montilivi s/n, 17071 Girona, Spain
E-mail: rosa.peracaula@udg.edu
Fax: 134-972418150
Abbreviations: ACT, a1-antichymotrypsin; AT, a1-antitrypsin;
ATIII, antithrombin III; EPO, erytropoietin; HPT, haptoglobin;
HPTb, haptoglobin b chain; pI/SA, pI units per sialic acid
molecule; RNase 1, human pancreatic ribonuclease; SA,
sialic acid
O-glycan chains, the pI of the protein is also changed. SA
refers to a large family of charged sugars, and among the
different types of SAs, 5-N-acetyl neuraminic acid is the
most common component of the SA family in N-linked
mammalian glycoconjugates.
The knowledge of how each type of PTM alters protein
2-DE migration patterns will aid in their interpretation. Since
this goal cannot be achieved easily, each PTM should be
studied separately in order to determine its influence before
all PTMs can be considered together. A complete and detailed
knowledge of how glycosylation modifies the behaviour of
proteins under isoelectrofocusing and PAGE conditions are
required in order to understand protein shifts in 2-DE.
Here, we have focused on the effect of sialylation on the
mobility of glycoproteins in 2-DE. Sialylation has important
roles in protein activity [7], blood half-life and hepatic
removal of serum glycoproteins [7, 8] and pharmacody-
namics [9]. It is also altered in pathological conditions such
as cancer [10–12]. These important functions have pushed
the scientific community to study sialylation, and much
information is already available in the literature, although so
far no specific studies about the effect of the SA on protein
pI have been performed.
In this study, we first reviewed the literature for
available information about the effect of SA on the pI of
These authors have contributed equally to this work.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
2904 S. Barrabés et al.
glycoproteins evaluated by 2-DE. We then examined the
influence of SA on the pI of three human serum glycopro-
teins with very different theoretical pIs: a1-antitrypsin (AT,
pI 5 5.35), haptoglobin b chain (HPTb, pI 5 6.32) and
human pancreatic ribonuclease 1 (RNase 1, pI 5 8.98). The
N-glycans of these proteins have been fully characterised
previously by N-glycan sequencing and MS [13–14].
The measurements of the pI shift and their relationship
with the SA content as well as the train of spots in the 2-DE
gels for HPTb and AT was analyzed in several human sera.
A linear correlation between the SA content of each of the
protein glycoforms and its pI was observed for HPTb, AT
and RNase 1. The pI shift induced by the presence of SA on
a glycoprotein was not the same among these proteins, and
increased with the pI of the protein.
2 Materials and methods
2.1 Serum samples
Sera from two healthy donors (C1, C2), five pancreatic
adenocarcinoma patients (N1–N5) and two chronic pancrea-
titis patients (P1, P2) were obtained from the Hospital
 
ð1  % N  1 SAÞ1ð2  % N  2 SAÞ1ð3  % N  3 SAÞ1ð4  % N  4 SAÞ
SA content ¼
400
 
ð1  % O  1 SAÞ1ð2  % O  2 SAÞ
1  Max O  SA
Universitari Dr. Josep Trueta de Girona (Girona, Spain)
with the approval for their use from its Ethics Committee.
2.2 2-DE of serum samples
2-DE was carried out as described previously [13]. First-
dimension IPG strips were focused in an IPGphor II
(Amersham Biosciences) and electrophoresis was
performed in an Ettan Dalt unit. Images were captured on
a Molecular Imager GS-800 Calibrated Densitometer
(BioRad, Hercules, CA, USA).
2.3 Amino terminal sequencing
Amino terminal sequence analyses were performed by
automated Edman degradation on an Applied Biosystems
Procise 492 cLC at the Proteomics Platform of Parc Cientı́fic
de Barcelona (Spain).
2.4 RNase 1 sample preparation and 2-DE
Serum from a pancreatic adenocarcinoma patient (N5) with
high levels of RNase 1 was purified by several chromato-
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2010, 31, 2903–2912
graphic steps as described previously [15]. 2-DE was
performed as described previously [14]. Strips were focused
on a Multiphor II (GE Healthcare), and the second dimension
was carried out on a horizontal 8–18% gradient SDS-PAGE
(GE Healthcare). Separated proteins were transferred onto a
PVDF membrane, and RNase 1 was detected with specific
antibodies, as described previously [10].
2.5 pI assignment
In order to enhance the accuracy of pI assignment, the strip
length was measured each time for each strip, and the
distance from the middle of the spots to the anodic edge was
also measured. The pI assignment was performed by
transforming the spot distance into a pI value using the
‘‘pI-distance on strip’’ function supplied by the manufac-
turer of the strips.
2.6. SA content calculation
The determination of the content of SA on a given glycoform
was calculated according to [16] using the following formula:
 Max N  SA
ð1Þ
200
where %N  1SA corresponds to the percentage of mono-
sialylated N-linked structures, %N  2SA corresponds to the
percentage of disialylated N-linked structures, and so forth.
Equally, %O  1SA and %O  2SA corresponds to the percen-
tage of mono- and disialylated O-linked structures. These
percentages are normalised by the denominator, 4  100 for
N-linked structures and 2  100 for O-linked structures. Max
N  SA and Max O  SA stand for the maximum number of SA
molecules in N-linked and O-linked structures, respectively,
taking into account that each N-glycosylation site can contain
up to four SA molecules, and each O-glycosylation site can
contain a maximum of two SA molecules. When estimating
the SA content from bibliographic data, the same formula was
applied whenever possible.
3 Results and discussion
3.1 State of the art
The detailed effects of SA on the pI of glycoproteins and the
corresponding shifts in mobility during 2-DE experiments
have not been studied in detail, although the influence of SA
on glycoproteins has already been reported [17–21]. So far,
only a few studies have paid attention to the direct influence
of SA on the pI shift of a glycoprotein [19–20] (Table 1).
www.electrophoresis-journal.com
Electrophoresis 2010, 31, 2903–2912 Proteomics and 2-DE 2905

Table 1. Bibliographic summary of glycoproteomic studies with relevant information about the SA content on glycoforms separated by
2-DE and its influence on the pI

Reference Protein Swiss-Prot no. Mw (kDa) pI (unglyc) N-glyc sites O-glyc sites Max. SA content pI decrease per SAa)

Schlags RhEPO P01588 18 8.75 3 1 14 0.24–0.32

Kremser ATIII P01008 49 5.95 4 – 16 0.07
Plematl ATIII P01009 49 5.95 4 – 16 0.07
Wilson HPT P00738 27 6.32 4 – 16 0.21
ACT P01011 45 5.32 2 – 8 0.24
AT P01009 44 5.37 3 – 12 0.31
a2-HS-glycoprotein P02765 37 5.27 2 3 14 0.31
He HPT P00738 27 6.32 4 – 16 0.16
Holland k-Casein variant A P02668 19 5.93 – 6 12 0.18
k-Casein variant B 19b) 6.34b) – 5 10 0.21

a) pI shift per SA molecule.

b) Calculated using Expasy compute pI/Mw tool (http://ca.expasy.org/tools/pi_tool.html) in base of k-casein variant A after substituting
T157-I and D169-A, which leads to variant B.

Antithrombin III (ATIII), which acts as a potent inhi-
bitor of blood coagulation, is a glycoprotein with four
N-glycosylation sites that presents two isoforms (ATIIIa and
ATIIIb) differing in one carbohydrate side chain on Asn-135
that is present in ATIIIa but not in ATIIIb. ATIII isoform a
contains four carbohydrate side chains, one more than
isoform b, which consist of a biantennary di-sialylated
complex type chain. Both isoforms have pIs lower than 6,
with reported values of 5.18 and 5.315 [19]. The pI difference
between the isoforms is about 0.14 pH units, and it is due to
the extra glycan chain with two SA units in isoform a. Thus,
each SA contributes to a decrease in pI of 0.07 pH units [19].
This was confirmed by another study [20] through glycan
analysis of the four glycopeptides of ATIII previously sepa-
rated by IEF. Moving from the most acidic to the most basic
glycoform, each contains one SA less indicating a decrease
of 0.07 pI units per SA molecule [20].
Erythropoietin (EPO) is a sialoglycoprotein of 166 amino
acids with three N-glycosylation and two O-glycosylation sites.
Many authors have studied the glycosylation features of
human and recombinant EPOs in order to distinguish them
due to both the misuse of rEPOs among athletes and to the
clinical use of these drugs as a treatment for anaemia [16, 18,
22, 23]. Several spots corresponding to rEPOs are resolvable at
pIs between 3 and 5, depending on the source [22–25].
Different studies have concluded that the trains of spots
observed in 2-DE for EPO (both human and recombinant) are
a result of different sialylation patterns of EPO, but none of
these studies have obtained a correlation between the pI and
the SA content [16, 18, 22, 23]. According to one study [18], the
total average content of SA of rEPO (Neorecormons) is
13 mol/mol EPO, and the pI shift after sialidase treatment is
approximately 3.1–4.1 pH units. Thus, the contribution of SA
to this pI shift can be calculated as 3.1/13 pI units per sialic
acid molecule (pI/SA) – 4.1/13 pI/SA 5 0.24–0.32 pI decrease
per SA molecule. Full characterisation of the SA content of
each rEPO spot resolved by 2-DE has already been performed
[23], but the pI was not assigned to each spot making it
impossible to calculate the correlation between the SA content
and the pI.
k-Casein, one of the major proteins in the milk, is a
glycoprotein of 19 kDa with six O-glycosylation sites and two
known phosphorylation sites. Among the known variants,
micro-heterogeneity of variant A (unmodified k-casein) and
variant B (differing by two amino acid substitutions that
result in the loss of one of the O-glycosylation sites) has
been studied by 2-DE combined with PMF [26]. Both
variants present one or two phosphorylated residues and
only forms with one phosphate appear to contain glycan
structures with up to six SA molecules. Taking into account
only the pI of the forms containing glycan structures (all of
them contain one phosphate group), the effect of the SA on
the pI of the glycoprotein can be calculated to be 0.18 pI
decrease per SA molecule for variant A and 0.21 pI decrease
per SA molecule for variant B.
N-Glycans from serum haptoglobin (HPT), a1-anti-
chymotrypsin (ACT), and AT and a2-HS-glycoprotein
separated by 2-DE have been fully characterised in a
previous study [27]. In that study, proteins from serum
samples were separated by 2-DE, and their glycan structures
from the spots of interest were characterised by LC-ESI-MS.
The glycan abundance in each structure was determined for
each pI isoform from each protein. According to the results,
the SA content for each protein permitted the calculation of
0.21 pI decrease per SA molecule for HPT, 0.24 pI decrease
per SA molecule for ACT, 0.31 pI decrease per SA molecule
for AT and 0.31 pI decrease per SA molecule for a2-HS-
glycoprotein. Serotransferrin was also analysed in the study,
but for this protein, the average sialylation increased with
increasing pI of the isoforms. The authors speculated that
the reason for this unexpected result could be other PTMs or
oligosaccharide site occupancy.
The influence of SA content on the pI of HPT can also
be deduced from previously reported results [21]. HPT was

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
2906 S. Barrabés et al. Electrophoresis 2010, 31, 2903–2912

separated by 2-DE, the major glycoforms were characterised
by normal-phase HPLC and the SA abundance was deter-
mined for each pI isoform. According to these results, and
calculating the SA content as above, the pI shift due to the
presence of a SA was a decrease of 0.16 pI units per SA
molecule, which is lower than the result deduced from the
previously commented study [27].
Many other proteins have been analysed in proteomic
studies, but it is difficult to obtain a correlation between the
SA content and the pI since both pieces of information are
not always available. Thus, we have performed the 2-DE
separation of three different serum proteins (HPTb, AT and
RNase 1) to obtain this correlation and compare it with the
correlations deduced from the existing literature in order to
clarify whether there is a common influence of SA on the
glycoprotein pI or whether the pI shift depends also on
other parameters such as the protein pI, type of glycan
structure, molecular mass or similar parameters.
All the studies reviewed above stem from 2-DE infor-
mation. In order to understand SA influence on the position
of glycoprotein spots, it is important to use a sensitive and
quantitative method that allows the analysis of the spots
excised from the gel. The glycan data for the HPT, AT and
RNase 1 used in this study were obtained by normal-
phase HPLC methodology [28], which is suitable for deter-
mining both neutral and charged monosaccharides, detects
up to femtomole levels of sugars and allows accurate
quantitative measurements of the amounts of individual
glycans.
3.2 HPTb and AT: Models of highly abundant
proteins
Many of the more abundant proteins in serum are
glycoproteins, such as HPT and AT. HPT is a tetramer of
two a and two b chains (Swiss-Prot No. P00738). This
structure is disrupted under the 2-DE conditions that resolve
these chains separately. Only the b chain contains glycan
structures, in particular it has four N-glycosylation sites. AT

Figure 1. An outline of the

experimental flow to study
the relationship between the
SA content and the pI of HPTb,
AT and RNase 1 from serum
samples. (A) 2-DE of serum
samples (Coomassie staining)
where HPTb and AT are
circled and numbered, (B)
2-DEs of RNase 1 purified
from serum (Western blot):
high glycosylated (B.1),
medium glycosylated (B.2)
and low glycosylated (B.3)
fractions. RNase 1 spots are
marked with the number of
SAs, referred to as S.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Electrophoresis 2010, 31, 2903–2912
is a serine protease inhibitor with three N-glycosylation sites
(P01009).
As shown in Fig. 1, serum samples from two healthy
people, four pancreatic adenocarcinoma patients and two
chronic pancreatitis patients were separated by 2-DE and
their glycan structures were determined [13]. The assign-
ment of these proteins was performed by MALDI MS and
MS/MS [13]. No other PTMs apart from N-glycosylation
were detected. Since the presence of some N-terminal
truncated forms of serum AT has been reported previously
[17, 29], AT spots were subjected to N-terminal sequence
analysis and contained the mature protein, with a regular
NH2 terminus.
HPTb and AT contain only complex type structures,
mainly bi- and tri-antennary but also tetra-antennary
glycans, with different numbers of SA moieties (from 1 to 4)
depending on the spot, and some external fucoses. During
2-DE, glycoforms of HPTb and AT separate as a train of
spots with similar molecular weights (about 40 kDa for
HPTb and 49 kDa for AT), but with different pIs (Fig. 1,
gel a). HPTb has six main spots, with pIs from 4.9 to 5.6
(named HPT1 to HPT6 from more acidic to more basic);
and AT separates into three spots, with pIs from 4.9 to 5.2
(named AT1 to AT3 from more acidic to more basic). The
glycan structures of each HPTb and AT isoform for these
eight different sera samples have been described previously
(by normal-phase HPLC, exoglycosidase digestions arrays
and MS) by our group [13].
From the 2-DE, each spot was excised from the gel and
the proportion of mono-, di-, tri- and tetra-sialylated struc-
tures were unequivocally assigned [13]. These determined
N-glycan structures allowed us to calculate the SA content
for each of the spots of HPTb and AT from all serum
samples as described in Section 2. Regarding this calcula-
tion, an integer number of SA molecules/protein molecule
can only be achieved when the N-glycan composition from a
particular spot contains molecules with the same degree of
site occupancy. Both AT and HPTb contain more than one
occupied N-glycosylation site and in a single spot, there is
likely to be a mixture of glycoforms, each one containing the
same number of SAs but distributed in different sites and/
or different structures. Thus, the methodology only permits
to calculate an ‘‘average’’ SA content considering the
percentage of mono-, di-, tri- and tetra-sialylated forms, and
in this case the result leads to a noninteger number of SA.
From these data, we investigated whether a relationship
between the pI of each spot and the SA content could be
established and a negative linear correlation was found for
both proteins from all eight serum samples (Fig. 2),
although the pI shift per SA molecule (represented by the
slope as pI decrease per SA molecule) was different for
HPTb and AT. Table 2 summarises the SA content and
glycoforms pIs from all eight serum samples, as well as the
slope: pI decrease per SA molecule, and the correlation (r)
between these two parameters. The effect of SA on the pI of
each protein was calculated as the median value of all eight
samples and was 0.16 pI decrease per SA molecule (SD 0.02)
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics and 2-DE 2907
for HPTb, and 0.13 pI decrease per SA molecule (SD 0.03)
for AT. After checking the normality and variance homo-
geneity of the data, the T-Test was carried out to compare
the pI decrease per SA molecule value obtained for HPTb
and AT, and these were found to be significantly different
(p 5 0.007).
Although the pI shift observed for HPTb is lower than
one previously observed [27] (0.21 pI decrease per SA
molecule), it is exactly the same as that calculated with the
results from another study [21]. The pI shift per SA for AT in
our study is considerably lower than the one calculated from
Wilson et al. [27] (0.31 pI decrease per SA molecule). As will
be discussed later, all the data calculated using the results
from Wilson et al. [27] seem to differ in comparison with the
other references and the present data.
The pI shift per SA for both HPTb and AT has also been
estimated theoretically considering the protein sequence
(considering only the chain described in Uni-Prot Base), the
pKa values of the dissociable amino acids (pKa values after
Bjellqvist et al. [30]) and the pKa of the SA (which is 2.6)
using a bioinformatic tool from the Friedrich Miescher
Institute for Biomedical research (http://www.fmi.ch/
members/reto.portmann/pepeval.html). The amount of SA
could be up to 16 SA molecules per HPTb molecule (four
N-glycosylation sites) and up to 12 SA molecules per AT
molecule (three N-glycosylation sites). The values of the
theoretical pI shift showed a different tendency for each
protein. In the case of HPTb, there is a shift of 0.073 pI
units per SA molecule when adding the first four SA units
(theoretical pI of the protein between 6.32 and 6.04), but the
tendency drops to 0.031 pI decrease per SA molecule when
adding from 5 to 16 SA units (theoretical pI of the partly
sialylated protein between 5.98 and 5.63). In the case of AT,
the pI shift tendency is constant when adding from 0 to 12
SA units, which is 0.011 pI decrease per SA molecule
(theoretic pIs between 5.4 and 5.26). These results seem to
indicate that an additional negative charge does not have a
great influence in the pI of a negative protein (such as AT)
and the influence increases as more basic the protein is
(such as the different tendency for unsialylated HPTb and
highly sialylated HPTb). These estimations are lower than
the empirically observed values (in this study and the
reviewed ones), but indicates that the SA causes a major
decrease in pI as less acid the protein is.
3.3 RNase 1: A model of a low abundance protein
Ribonuclease 1 (RNase 1) is a glycoprotein with three
N-glycosylation sites that have also been characterised
previously by N-glycan sequencing [10, 14]. It is present at
very low levels in serum and cannot be directly detected after
2-DE of serum using Coomassie staining. This is the reason
why the approach for analysing the SA content in the RNase
1 differed from that of HPTb and AT (Fig. 1). In this case,
the protein was purified in order to obtain the glycoforms
with one, two and three N-glycosylation sites occupied. The
www.electrophoresis-journal.com
2908 S. Barrabés et al. Electrophoresis 2010, 31, 2903–2912

A 5.8 Haptoglobin
5.2
Antitrypsin
5.7
5.6 5.2
5.5
5.1
5.4
pI

5.3

pI
5.1
5.2
5.1 5.0
5.0 5.0
4.9
4.8 4.9
6.00 7.00 8.00 9.00 10.00 11.00 6.00 6.50 7.00
SA content SA content

C1 C2 N1 N2 N3 N4 P1 P2 Median SD

pI/SA 0.17 0.16 0.14 0.15 0.19 0.15 0.17 0.15

HPT β 0.16 0.02
r -0.97 -0.99 -0.99 -0.98 -1.00 -0.99 -0.99 -0.99
pI/SA 0.17 0.09 0.13 0.11 0.12 0.16 0.12 0.10
AT 0.13 0.03
r -0.98 -1.00 -1.00 -0.98 -0.96 -0.99 -1.00 -0.99

B 9.5 RNase 1 Figure 2. Correlation between the

SA content and the pI of HPTb
9 (A left) and AT (A right) glycoforms
from eight different serum samples
8.5 ∇ 0.28 pI/SA High glyc.
and of RNase 1 glycoforms from a
pI

Medium glyc.
8 Low glyc. serum sample (B). The pI shift per
SA is obtained from the slope of the
7.5 linear regression obtained by plot-
ting the SA content against the pI of
7 each glycoform. The median and
0 1 2 3 4 5 6 SD (for HPTb and AT) are also
Estimated SA content presented.

glycan composition and SA content of each of these forms is
already known [14].
Thus, RNase 1 from a pancreatic cancer patient’s serum
(with a high RNase 1 concentration) was completely purified
as described previously [14]. The pure RNase 1 fractions
were pooled according to their mobility during SDS-PAGE
into pools with high, medium and low glycosylation levels
(Fig. 1). Highly glycosylated forms contain RNase 1 with the
three N-glycosylation sites occupied, medium glycosylated
forms correspond to the RNase 1 with two N-glycosylation
sites occupied and low glycosylation forms contain the
RNase 1 forms with just one N-glycosylation site occupied,
according to prior work [15]. High, medium and low
glycosylated RNase 1 fractions were run separately in three
2-DE gels, and a train of spots was obtained for each of them
(Fig. 1B).
The major glycan structures described for serum RNase 1
are mono- and disialylated bi-antennary glycans. Each
N-glycosylation site can contain one or two SAs linked to the
terminal end of the complex biantennary structure (Fig. 3).
Thus, the most probable glycan structure on the high, medium
and low glycosylated RNase 1 permits to estimate the SA
molecules/RNase 1 molecule in each RNase 1 spot and
correspondingly the estimated SA content of each spot. The
major RNase 1 forms with three N-glycosylation sites occupied
could have four or five SA residues, and these could be
assigned to two spots at pIs 7.61 and 7.31 detected after
2-DE of the high glycosylated RNase 1 (Fig. 2B left). Similarly,
the major forms of RNase 1 with two N-glycosylation sites
occupied could have two, three or four SA residues and were
assigned to the three most intense spots at pIs 8.26, 7.95 and
7.65 for medium glycosylated RNase 1 (Fig. 2B centre). Finally,
the low glycosylated RNase 1 can contain only one or two SA
residues, and the corresponding spots were detected at pIs 8.38
and 8.24 (Fig. 2B right). These results allow the establishment
of a negative linear correlation (decrease of 0.28 pI units per SA
molecule, r 5 0.99) between the SA content and the pI of the
protein (Fig. 2).
2-DE of complex samples from conditioned media of
different cell lines containing RNase 1 [14] showed a train of
spots corresponding to the sum of the spots of the pure
RNase 1 separated into high, medium and low glycosylated
fractions. However, the SA content of each of these sample
spots could not be determined unequivocally, and hence it
was not possible to calculate the correlation between the pI
and the SA content for these other samples.
The theoretical pI shift per SA for RNase 1 has also been
estimated as in the case of HPTb and AT, taking into
account that the amount of SA could be up to 12 SA
molecules per RNase 1 molecule (three N-glycosylation

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 Table 2. SA content and values of pI decrease per SA molecule calculations

C1a) C2a) N1a) N2a) N3a) N4a) P1a) P2a) All samples

SA contentb) pIc) SA contentb) pIc) SA contentb) pIc) SA contentb) pIc) SA contentb) pIc) SA contentb) pIc) SA contentb) pIc) SA contentb) pIc) SA contentd) SDd) pId) SDd)

HPT1 9.91 4.93 10.44 4.93 10.77 4.98 10.59 4.96 10.08 4.93 9.90 4.87 10.67 4.94 10.76 4.96 10.39 0.37 4.94 0.03
HPT2 9.50 5.01 10.15 5.01 9.74 5.07 9.61 5.04 9.59 5.02 9.43 4.95 9.98 5.02 9.97 5.04 9.75 0.26 5.02 0.04
HPT3 8.92 5.12 9.08 5.10 8.86 5.17 8.88 5.14 8.94 5.11 8.69 5.04 9.28 5.12 9.26 5.14 8.99 0.20 5.12 0.04
HPT4 8.10 5.24 8.38 5.22 8.10 5.30 8.19 5.25 8.14 5.24 8.01 5.16 8.69 5.24 8.53 5.25 8.27 0.24 5.24 0.04
HPT5 7.36 5.38 7.83 5.35 7.39 5.42 7.43 5.37 7.46 5.37 7.48 5.29 8.17 5.37 8.12 5.38 7.65 0.33 5.37 0.04
HPT6 7.52 5.50 6.71 5.48 6.61 5.54 7.17 5.48 6.89 5.50 7.06 5.42 7.36 5.49 6.99 5.50 7.04 0.31 5.49 0.03
Electrophoresis 2010, 31, 2903–2912

pI decrease 0.20 0.15 0.14 0.15 0.17 0.19 0.17 0.15 0.16
/SA
R 0.97 0.99 0.99 0.98 1.00 0.99 0.99 0.99 0.99
AT1 6.77 4.99 7.16 4.99 6.91 5.04 6.84 5.01 6.83 4.99 6.67 4.91 6.91 4.99 6.98 5.03 6.88 0.15 4.99 0.04
AT2 6.48 5.03 6.71 5.03 6.60 5.09 6.59 5.05 6.63 5.04 6.47 4.96 6.64 5.03 6.71 5.07 6.60 0.09 5.04 0.04
AT3 6.25 5.08 6.23 5.08 6.12 5.14 6.01 5.10 6.11 5.08 6.09 5.01 6.17 5.09 6.15 5.12 6.14 0.08 5.09 0.04

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

pI decrease 0.17 0.09 0.13 0.11 0.12 0.16 0.12 0.10 0.13
/SA
R 0.99 1.00 1.00 0.98 0.96 0.99 1.00 0.99 0.99

a) C refers to sera from control patients, N refers to sera from pancreatic neoplasia patients and P refers to sera from pancreatitis patients.
b) Value obtained using the formula (1) described in Section 2.
c) Spot pI is given with two digits behind the coma in order to make clearer that the spots are in different strip positions.
d) Mean values (SA content or pI) and SD of data from the eight different serum samples (left).
Proteomics and 2-DE

www.electrophoresis-journal.com
2909
2910 S. Barrabés et al. Electrophoresis 2010, 31, 2903–2912

Observed pIs in 0.4

High glycosylated RNase 1
1+1+1=3 N5
0.35
1+1+2=4 EPO_Schlags
AT_Wilson AHSG_Wilson
+1 SiaAc 1+2+1=4 0.3
+2 SiaAc 4 SiaAc pI=7.61
1+2+2=5 RNasa
+1 SiaAc 0.25 AC_Wilson
2+1+1=4 kCAS-B_Holland

pI / SA
+2 SiaAc 5 SiaAc pI=7.31 HPT_Wilson
+1 SiaAc 2+1+2=5 0.2
+2 SiaAc kCAS-A_Holland
2+2+1=5 HPT_He
2+2+2=6 0.15
HPT
AT
Medium glycosylated RNase 1 0.1
ATIII_Plematl
1+1=2 2 SiaAc pI=8.26 0.05
+1 SiaAc
+2 SiaAc 1+2=3
0
3 SiaAc pI=7.95
4 5 6 7 8 9 10 11
+1 SiaAc 2+1=3 pI (unglycosylated protein)
+2 SiaAc
2+2=4 4 SiaAc pI=7.65
Figure 4. The values of pI decrease per SA molecule obtained in
Low glycosylated RNase 1 this study and the reviewed works versus the theoretical pI of
+1 SiaAc 1 SiaAc pI=8.38 unglycosylated proteins; the results of the linear fitting are
+2 SiaAc 2 SiaAc pI=8.24 shown. Results from works excluded from the correlation
analysis are shown in grey.

Figure 3. Theoretical proportion of SA content in RNase 1 with

three, two and one (from top to bottom) N-glycosylation sites
occupied by mono- or disialylated bi-antennary structures. The
pIs of the major spots of pure serum RNase 1 shown in Figs. B1,
B2 and B3 are presented.
sites). The theoretical pI shift is 0.29 pI decrease per SA
molecule, and it has a constant tendency along the SA
addition. The estimation is practically the same than the
empirically observed value in this work (0.28 pI decrease per
SA molecule), which indicates again that the additional
negative charge has a great influence in basic proteins.
As only one RNase 1 sample could be fully analysed, no
statistical comparison of the pI decrease per SA molecule
value with those determined for HPTb and AT was
performed. The obtained RNase 1 value of pI decrease per
SA molecule (0.28) is, however, notably higher than the
values obtained for HPTb (0.1670.02) and AT (0.1370.03).
3.4 Acidic proteins versus basic proteins
The influence of the SA on the protein pI shows a linear
correlation, although it varies for each of the glycoproteins
studied (HPTb, AT and RNase 1). One of the main
differences between these glycoproteins is their theoretical
pI based on their amino acid composition. Considering the
other proteins reviewed herein, we can establish two groups
of proteins: acidic proteins such as HPTb (pI 5 6.32), AT
(pI 5 5.35), ATIII (pI 5 5.95), ACT (pI 5 5.32), a2-HS-
glycoprotein (pI 5 5.27) and k-casein (pI 5 5.93); and basic
proteins such as RNase 1 (pI 5 8.98) and EPO (pI 5 8.75),
summarised in Table 1. As commented above, the tendency
observed for these proteins is a greater decrease in pI per SA
molecule with increasing glycoprotein pI. Roughly speak-
ing, glycoproteins with acidic pIs show a pI decrease of
about 0.1–0.2 units per SA, whereas basic glycoproteins
show a decrease of 0.22–0.3 pI units per SA. Some of the
correlations of pI decrease per SA calculated according to
bibliographic data seem to show a bias. In particular,
Plematl et al. [20] calculated a very low decrease in pI per SA
when studying ATIII (0.07 pI decrease per SA molecule), but
no possible comparison can be made in this case since no
other studies about SA content and its influence on the
ATIII pI could be found. In addition, the study of Wilson
et al. [27] showed relatively higher values of pI decrease per
SA molecule for all the glycoproteins studied than the values
calculated from other literature sources or this study. In fact,
only the pI shift per SA of HPT in Wilson et al. [27] is in the
range of acidic proteins (decrease of 0.1–0.2 pI units per SA),
but it is still higher that the HPTb values determined in this
study and in He et al. [21]. Thus, all the calculated values of
pI decrease per SA molecule from Wilson et al. [27] seem to
be overestimated, which might be due to insufficient
quantitative detection of the amounts of SA.
Considering only the data from this study and the
reviewed articles of which we have no reservations about the
SA detection, there is a linear positive correlation
(slope 5 0.04, r 5 0.90) between the protein pI and the pI
shift induced by the presence of SA (Fig. 4). Thus, it seems
that the influence of SA on glycoprotein pI becomes more
marked in more basic proteins, which can also be observed
by an in silico approach using the modelling bioinformatic
tool.
Larger changes on pI decrease upon the addition of a
negative charge on basic proteins compared with acidic
proteins, which undergo smaller changes on pI decrease,
have also been observed when considering the effect of
phosphorylation on the pI shifts of different proteins [31].
These findings are not surprising: pI is defined as the
pH at which all the charges in a protein sum to zero. The
charge of the protein is equivalent to the sum of the frac-
tional charges of the protein’s charged groups (N-terminal,
C-terminal, K, R, H, D, E, C and Y), taking into account the
number of each amino acid residues from the protein
sequence. The proportion of chemical groups that take on a
charge in response to pH is given by their pKa values and
their difference with the pH value (10pKapH for positive

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Electrophoresis 2010, 31, 2903–2912
groups and 10pHpKa for negative groups). SA group has a
pKa of 2.6. In proteins rich with basic amino acids (high pI),
the addition of a SA results in a strong negative charge at
pHs close to its pI. The corresponding gain of positive
charges to get a net electrical charge requires a high pH
decrease, which explains why the pI of the resulting protein
is much lower. On the other hand, when a protein has a
higher proportion of acidic amino acids (low pI), the addi-
tion of a SA will result in weak negative charge (at the pHs
close to its pI). In this case, only a weak pH decrease is
required to achieve a neutral global charge, which explains
that the resulting protein will have a slightly pI decrease
compared with the unmodified protein.
Despite the accordance in the tendency between the
theoretical and the empiric results, it should be taken into
account that the value of pI decrease per SA molecule esti-
mated using the modelling tool matches up with the
empirically observed value in the case of a basic protein
(RNase 1), but it is much lower than the empiric one in the
case of acidic proteins (HPT and AT). This divergence may
be somehow attributed to several aspects not considered in
the theoretical approach, such as the glycoprotein environ-
ment involved in the ionization state of the amino acids,
which influence the spots position. It is known that the pKa
of a charged amino acid residue can be affected by the
spatial distribution of surrounding charged residues. This is
a mechanism widely used by enzymes to keep a charged
amino acid catalytic residue in a protonated form. For
example, the glutamic acid 134 from a b glucanase works
sequentially as an acid base catalytic residue being initially
protonated, at pHs that it should not be, because it is
shielded by a ring of negative charged residues [32]. Rings of
hydrophobic residues can also provide the shield that keeps
residues uncharged, protonated, etc. Therefore, the 3-D
distribution of residues can be, to some extent, responsible
for differences between predicted and experimental pI.
4 Concluding remarks
Packer already stated in 1998 that, ‘‘the primary goal for
analysing the carbohydrate content of glycoprotein spots is
to understand the rules which govern the migration of
glycoproteins in 2-D electrophoresis’’ [17]. The key to
understanding these rules is to be able to produce predictive
vectors that would allow us to interpret changes in
glycoprotein patterns. The authors insisted on the impor-
tance of understanding the reason why glycoforms separate
into discrete spots during 2-DE rather than a smeared band
along the gel in order to comprehend the separation rules
for predictive analysis.
Literature about sialylation and its direct influence on
the 2-DE pattern of a glycoprotein has been reviewed.
Microheterogeneity in pI is attributed in many of these
studies to the SA content of the different glycoforms.
Unfortunately, although the SA content of each of these
glycoforms is known in many cases [23, 33], no correlation
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics and 2-DE 2911
between the SA content and the pI has been established so
far. Taking into account the hard task of characterising the
glycan structures and monosaccharide content of glycopro-
teins previously separated by 2-DE, it would be extremely
helpful if laboratories performing these type of analyses
could show the pI of each described glycoform. Therefore,
more data about the effect of SA content in glycoprotein
migration during 2-DE would be available and could be
added to the results presented here.
As shown in this study, correlation between the pI of
glycoproteins and the SA content appears to be linear, with a
contribution of 0.16 pI decrease per SA molecule (SD 5 0.02)
for HPTb, 0.13 pI decrease per SA molecule (SD 5 0.03) for
AT and 0.28 pI decrease per SA molecule for RNase 1. The
decrease in pI of a protein due to SA is not necessarily the
same for all glycoproteins. It could depend on the glycan
structure containing the SA residue or the presence of other
PTMs, but SA residues seem to, in general and according to
our data, have stronger effects on the pI of basic glycopro-
teins than that of acidic ones.
This work was supported by the Department of Science
and Technology from the Ministerio de Educación y Ciencia
(BIO 2004-0438 and BIO 2007-61323) and by the Foundation
La Marató de TV3 (grant 050932), awarded to R. P., the
Government of Catalonia (grant 2005SGR00065) awarded
to R. L., and P. M. R. is funded by the National Institute for
Bioprocessing Research and Training, Ireland.
The authors declare that they have no proprietary, financial,
professional or other personal interest of any nature or kind in
any product, service or company that could be construed as
influencing the position presented in this manuscript.
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