CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY
OF ZEIN PROTEINS FROM CONVENTIONAL AND TRANSGENIC MAIZE
Guillaume L. Erny a, Maria Luisa Marina b and Alejandro Cifuentes a,*
a Institute
b Department
of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
of Analytical Chemistry, Faculty of Chemistry, University of Alcalá, Ctra. MadridBarcelona Km. 33.600, 28871 Alcalá de Henares (Madrid), Spain.
*Corresponding author: Dr. Alejandro Cifuentes, Fax#: 34-91-5644853, e-mail: acifuentes@ifi.csic.es
Abbreviations: DMA, N,N-dimethylacrylamide; EpyM, ethylpyrrolidine methacrylate;
Keywords: Bt corn, CE-MS, capillary coating, food analysis, protein separation, GMO
1
Abstract
In this work, an original capillary electrophoresis-mass spectrometry (CE-MS) method has been
developed to analyze the complex zein proteins fraction from maize. A thorough optimization of i) zein
proteins extraction, ii) CE separation and iii) electrospray-mass spectrometry (ESI-MS) detection is
carried out in order to obtain highly informative CE-MS profiles of this fraction. The developed CE-MS
method provides good separation of multiple zein proteins based on their electrophoretic mobilities as
well as adequate characterization of these proteins based on their relative molecular mass (Mr). Zein
proteins with small Mr differences (below 100 Da) were easily separated and successfully analyzed by
CE-MS. Thus, apart of the so-called 15-kDa--zein and 16-kDa--zein, which are demonstrated to be
formed by a heterogeneous group of proteins, numerous -zeins belonging to the 19 and 22-kDa fraction
were also identified for the first time in this work. The usefulness of this CE-MS method was
corroborated by comparing the zein-proteins fingerprint of various maize lines including transgenic and
their corresponding non-transgenic isogenic lines cultivated under the same conditions.
2
1. Introduction
The alcohol soluble storage proteins (prolamines) of corn are a mixture of polypeptides that constitute
about 50-60% of the total endosperm protein. These proteins are classified in different groups on the
basis of differences in their solubility and sequence [1-3] among which α-, - and -zeins are considered
the majority group. The function of these proteins is apparently to store nitrogen for the developing seed.
Besides, the multiple uses given to these proteins show their considerable interest in many different
fields [4].
The zein proteins composition is usually characterized by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) [5] based on their molecular weight (Mw). However, SDS-PAGE exhibits
poor resolution and low accuracy in Mw assignment. More recently, matrix-assisted laser desorption
ionization mass spectrometry (MALDI-MS) [6,7] has been used demonstrating that this procedure
provides a much better information in terms of mass accuracy. However, the complexity of these zein
fractions makes very difficult the direct analysis of all the proteins that can be found especially those at
low concentration.
High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have also been
used to separate zein proteins [8]. Thus, proteins can be separated by their affinity to a stationary phase,
by their pI or by their electrophoretic mobility. Although these approaches provide excellent resolution,
the absence of information concerning the relative molecular mass of the separated proteins is an
important limitation. In this regard, coupling liquid separation techniques (HPLC or CE) with mass
spectrometry can be an interesting strategy to overcome this limitation. Thus, CE-MS (or HPLC-MS)
allows combining the high separation power of modern analytical separation techniques with the
information about analytes relative molecular mass (or even fragmentation pattern) provided by mass
spectrometry. Numerous reviews dealing with the application of LC-MS [9,10] and CE-MS [11-14] to
3
protein analysis have been published in recent years. However, to our knowledge, neither LC-MS nor
CE-MS has been used to analyze zein proteins. This might be due to the high tendency of zein proteins
to form aggregates through disulfide and hydrogen bridge bonds. For this reason, most separations are
performed either under reducing condition or using surfactants (e.g., SDS) [8] especially in CE, making
highly incompatible the separation step with the subsequent MS analysis.
The aim of this work is to design the first CE-MS method reported so far for the analysis of zein
proteins. This is expected to provide a good separation based on the protein electrophoretic mobilities in
conjunction with an accurate determination of the various protein Mr. This manuscript is the following
of a previous work also carried out in our lab in which was shown that aggregatable zein proteins can be
separated in a very simple buffer (in this case MS compatible) [15]. CE-MS separation of various maize
cultivars both conventional and genetically modified will also be compared.
2. Experimental Section.
2.1 Chemicals.
Sodium hydroxide, formic acid and 25% ammonium were from Merck (Darmstad, Germany). Sodium
dodecyl sulfate and acetonitrile were from Fluka (Seelze, Germany), 2-propanol, HPLC grade, from
Sharlau (Barcelona, Spain) and 2-mercaptoethanol from Sigma-Aldrich (Steinheim, Germany). Water
was deionized with a Milli-Q system (Millipore, Bedford, MA, USA).
2.2 Samples.
The different varieties of conventional and transgenic maize were obtained from a field assay carried out
in Estación Experimental Agrícola Mas Badia in Tallada d'Empordà (Girona, Spain) using commercial
4
varieties. Namely, Aristis maize (wild type and its Bt11 transgenic variety), Tietar maize (wild type and
its Bt11 transgenic variety) and PR33P66 maize (wild type and its Bt11 transgenic variety). To prepare
these samples, the maize kernels (transgenic and conventional) were separately milled to a fine powder
using different grinders.
2.3 Coating procedure.
Before first use, all capillaries for CE-UV and CE-MS (see below) were conditioned using a 20 min
rinse with 0.1 M NaOH and 1% SDS in water. Every morning, and between runs, the capillary was
coated
using
an
aqueous
solution
of
0.1
mg/ml
of
ethylpyrrolidine
methacrylate-N,N-
dimethylacrylamide (EPyM:DMA) copolymer [16]. The following procedure was used; the capillary
was washed for 1 minute with 0.1M NaOH and 1% SDS in water, then 2 min with a solution containing
0.1 mg/ml of EPyM:DMA polymer in water, and then 5 min with buffer. During the washing procedure
the nebulizing gas was turned off in order to avoid any contamination with the polymer. The NaOHSDS washing allows not only to remove any proteins precipitates, [17] but also to clean the capillary
wall in order to obtain a reproducible coating between injections.
2.4 CE-UV.
For the initial CE-UV analysis (see below) a CE apparatus (P/ACE 5010 Beckman Instruments,
Fullerton, CA, USA) equipped with a UV-Vis detector was used. The CE instrument was controlled by a
PC running System GOLD software from Beckman. Bare fused-silica capillaries (50 μm i.d.) were
purchased from Composite Metals Service (Worcester, England). The detection and total length were 30
and 37 cm, respectively. The detection was performed at 200 nm. The sample was injected for 6 s at
3.5x103 Pa. Separation was performed at -20 kV. The BGE used for CE-UV was 40:20:2:38 (v:v:v:v)
acetonitrile:isopropanol:formic acid:water.
2.5 CE-MS.
5
A CE apparatus (P/ACE 5010 Beckman Instruments, Fullerton, CA, USA) equipped also with a UV-Vis
detector was used. The CE instrument was controlled by a PC running System GOLD software from
Beckman. An ion trap mass spectrometer (Esquire 2000; Bruker Daltonik, Bremen,Germany) equipped
with an orthogonal electrospray interface (model G1607A from Agilent Technologies, Palo Alto, CA,
USA) was used. Bare fused-silica capillaries (50 μm i.d.) were purchased from Composite Metals
Service (Worcester, England). The total and detection length (to the MS) of the used capillaries were 90
cm. Injections were made using N2 at 3.5x103 Pa for 45 s. Separation was performed at -20 kV. The
BGE used for CE-MS experiments was 40:20:2:38 (v:v:v:v) acetonitrile:isopropanol:formic acid:water.
Electrical contact at the electrospray needle tip was established via a flow of sheath liquid which
consisted of 50:50 (v:v) iso-propanol:water and delivered at a flow rate of 0.290 ml/h by a 74900–00–05
Cole Palmer syringe pump (Vernon Hills, IL, USA). The mass spectrometer was operated in the positive
ion mode, and was scanned in the 1200–2200 m/z range during separation. ESI-MS operating conditions
were optimized by adjusting the position of the capillary into the needle counter electrode as indicated in
section 3.2. The optimum nebulizer/drying gas conditions were: a pressure of 2.76 x 104 Pa and 4 L/min
of nitrogen at 150C. The instrument was controlled by a PC running the Esquire NT software from
Bruker Daltonics.
2.6 Zein proteins extraction.
Proteins from transgenic and conventional grounded maize lines were extracted by checking different
procedures based on Bean et al. [18] and Adams et al. [7]. The selected procedure consisted in, briefly,
mixing 50 mg of each maize flour with 1 mL of a buffer composed of 60:5:35 (v:v:v) ACN:2mercaptoethanol:water adding to this solution concentrated ammonium hydroxide till a final
concentration of 120 mM. The sample was vigorously shaken for 5 min, then centrifuged for 1 min and
the aqueous organic phase collected for further use. This extraction was repeated three times. Proteins
were then precipitated by adding acetone to a ratio of 8 volumes of acetone per 1 volume of extraction
buffer. After centrifugation, the supernatant was removed and the pellet dried with air before being re6
dissolved. The whole protein extract was dissolved in 200 L of 60:2:38 (v:v:v) acetonitrile: formic
acid:water adding to this solution ammonium hydroxide till a final concentration of 120 mM and then
analyzed by CE-UV and/or CE-MS. Zein proteins were kept frozen at –20 ºC until used.
3. Results and discussion
3.1 Optimization of zein extraction and CE separation.
All the optimization experiments regarding zein extraction and CE separation were done using zein
extracts from Aristis maize (wild type). The extraction methodology was based on the work of Bean et
al. [18], and Adams et al. [7]. Thus, Bean et al. [18] found that extraction (under reducing conditions) of
-zeins was achieved using 60-70% of ACN, whereas extraction of -, -, and -zeins was high using
40-60% ACN. Adams et al. [7] observed that the 27- kDa -zein was better extracted in a basic buffer
(i.e. containing 25 mM of ammonium or higher). For this work, three extraction buffers were tested:
namely, i. 40:5:55 (v:v:v) ACN:mercaptoethanol:water; ii. 60:5:35 (v:v:v) ACN:mercaptoethanol:water;
and iii. 60:5:35 (v:v:v) ACN:mercaptoethanol:water adding to this solution concentrated ammonium
hydroxide till a final concentration of 120 mM. The extraction yield of zein proteins was measured using
CE-UV with a capillary of 37 cm length and the conditions described below.
The CE separation of zein proteins was based on a previous work [15] in which it was shown that zein
proteins could be separated using an ammonium concentration gradient between the background
electrolyte (BGE) and the sample zone (by using either 120 mM ammonium hydroxide in the sample
zone and zero in the BGE or vice-versa). The separations were performed in a BGE composed of
60:2:38 (v:v:v) ACN:formic acid:water; while zein proteins, after extraction and precipitation, were
dissolved in 60:2:38 (v:v:v) ACN:formic acid:water adding 120 mM ammonium hydroxide. In the
mentioned work [15], no significant differences in resolution were observed between cationic coated
7
and bare fused silica capillaries when ammonium hydroxide was used in the BGE. This is not the case
when there is no ammonium hydroxide in the BGE, and this is believed to be due to interactions
between proteins and the silica surface. To avoid such interactions, EPyM:DMA dynamically coated
capillaries were used for this purpose [19]. Such coating is achieved by simply flushing the capillary
with the polymeric solution, providing a fast anodal eof which allows the separation of the zein proteins
in less than 6 minutes on a capillary of 37 cm length. If higher resolution is necessary, it is better to use
in the BGE 40% ACN plus 20% isopropanol instead of 60% ACN since this will reduce the eof velocity
by about 20% [15].
EPyM:DMA coated capillary showed good stability over successive run, and the capillary can be used
without any recoating procedure. It has been shown that the lowest RSD is obtained when the capillary
is washed between runs with the polymeric solution [19]. However, when testing the coating in CE-MS
numerous spikes were observed. This was believed to be due to the polymer leaching. It was observed,
that the amount of spikes was significantly reduced by first rinsing the capillary with a wash solution
made of 1% SDS, 0.1 M NaOH in water, and then with the polymeric solution. The washing solution
was efficient in removing the coating from the capillary, and thus a fresh polymeric layer was applied
prior to each run. Such washing procedure was also used when working in CE-UV.
The CE-UV electropherograms obtained with the three extraction buffers can be observed in Figure 1.
As it can be seen, when using 40 % ACN in the extraction buffer (Figure 1A), the protein amount is far
less than with 60% ACN, however, the same number of proteins seems to be extracted. At 60% ACN,
the presence or absence of ammonium does not make a big difference apart from the last groups of
peaks (around 7 min in Figure 1B and 8 min in Figure 1C). For the rest of the paper, the latest extraction
buffer will be used. It has to be noted that when using extraction buffer containing 120 mM ammonium
hydroxide, good results were also obtained injecting the sample as it is (i.e. without precipitation step),
showing that if ammonium in the sample zone is required to achieve good separation, this is not the case
8
of formic acid. However, even if the extraction buffer used for the remaining of this work contains
ammonium hydroxide, protein precipitation will still be used; this, firstly, to provide a cleaner sample
for the MS work, and secondly for safety considerations due to the high concentration of
mercaptoethanol in the extraction buffer.
The repeatability of the separation was measured using four consecutive runs. The average relative
standard deviation (RSDn=4) of the effective mobilities using the first twelve peaks (i.e. the group of
peaks migrating between 5 and 7 min in Figure 1C) was equal to 0.8%, whereas the average RSD on the
peak area was equal to 7.6%. Reproducibility of the extraction method was measured using four
consecutive extractions. The average relative standard deviation (RSDn=4) of the effective mobilities
using the first twelve peaks (i.e. the group of peaks migrating between 5 and 7 min in Figure 1C) was
equal to 1.3%, whereas the average RSD on the peak area was equal to 10.5% indicating that a
reproducible extraction was obtained under these conditions.
3.2 ESI-MS optimization.
Numerous parameters can be optimized in ESI-MS when working in CE-MS, including sheath liquid
composition and flow, dry gas flow and temperature, position of the capillary in the spray chamber, etc...
[20] To make easier this optimization a new approach is proposed in this work that ensures good
spraying conditions when working with an ion trap analyzer. In brief, a fixed number of ions that can be
accumulated into the ion trap is set and best spraying conditions are assumed when the lowest
accumulation time is achieved (e.g., while varying the position of the CE capillary into the ESI needle or
modifying any other parameter). The principle of this procedure is similar to the one proposed by Geiser
et al [21]. In their work [21], the optimum position of the CE capillary into the ESI needle is assumed
when the electrical current in the MS capillary is between 30 and 100 nA, indicating a good spraying
while avoiding any corona discharge. With our procedure, when the lowest accumulation time is
achieved, the current varied between 30 and 50 nA what seems to confirm the usefulness of this method.
9
During the optimization process, nebulizer pressure and dry gas flow were kept constant and equal
respectively to 2.76 x 104 Pa and 4.0 l/min. Parameters studied were composition and flow of the sheath
liquid, dry gas temperature and capillary voltage. For the MS optimization, a 60% ACN, rather than
40% ACN 20% isopropanol, BGE was used in order to achieve faster separations. Thus, different
solutions were tested as sheath liquid by mixing an organic solvent, namely, acetonitrile (ACN),
methanol (MeOH) and/or isopropanol, with water. No acid was used as the concentration of formic acid
in the BGE was already high (2%). Under these conditions, it was observed that good MS spectra were
obtained for all organic solvents at a given solvent:water ratio and sheath liquid flow. Indeed, using
several of the mentioned conditions, good S/N ratios were observed together with high CE resolution in
the total ion electropherograms (TIE). However, in many cases the MS spectra obtained were useless.
Examples are shown in Figure 2 with MeOH. In this Figure various TIE are shown (I) in parallel with
the MS spectra (II) of the peak marked with an asterisk. As can be seen in Figure 2, good MS spectra for
the selected peak (i.e., a protein with relative molecular mass (Mr) of 26634) were obtained using either
a 80:20 (v:v) MeOH:water sheath liquid and a flow of 0.24 ml/h (Figure 2A), or a 90:10 (v:v)
MeOH:water sheath liquid and a flow of 0.10 ml/h (Figure 2B). However, with a 80:20 (v:v)
MeOH:water sheath liquid and a flow of 0.10 ml/h, the MS spectra cannot be used since it is too noisy
(Figure 2C). However, as can be seen in the TIE, this is not due to bad electrospray conditions. Indeed,
the best signal to noise ratio (S/N) was obtained in this last case (electropherogram of Figure 2C),
whereas the cleanest MS spectra was observed for the TIE with the lower S/N ratio (see Figure 2B (I)
and (II)). No fully satisfactory explanation was found to explain the MS spectra observed in Figure 2C
(II). With MeOH, at lower percentages of organic solvent no peaks were observed. This was believed to
be due to protein precipitation, and was confirmed by the presence of a white powder in the spray
chamber under these conditions.
Effect of the dry gas temperature was also studied, and it was observed that the dry gas temperature has
no significant effect on the S/N ratio of electropherograms and their corresponding MS spectra. This can
10
be observed comparing Figure 2A (II) (dry gas temperature= 250C) and Figure 2D (II) (dry gas
temperature= 150C) in both the same sheath liquid delivered at 0.24 mL/h. It could also be observed
that although both MS spectra allowed an adequate determination of the protein Mr, a modification of
the number of charges on the protein was observed, increasing the average number of charge per protein
with the temperature decreasing. Thus, Figure 2A, done at 250 ºC, shows the most intense ion at m/z =
1776.8, while in Figure 2D, done at 150 ºC, the most intense ion is observed at m/z = 1665.7. This has
been further confirmed with an experiment done at 50 C (data not shown) showing the most intense
peak at m/z = 1567.6, corresponding to 17 charges. This result (that was demonstrated to be reproducible
in a triplicate experiment) is unusual since normally a shift toward higher m/z values is observed when
the temperature is decreased [22], which has been linked to a modification of the three-dimensional
structure of the protein (i.e. unfolding of the protein at higher temperatures). However, this explanation
is not consistent here, as this would indicate a protein folded tighter at 250ºC than at 50ºC. At the
moment, we do not have an adequate explanation for this result.
Both ACN and isopropanol could be used instead of MeOH. Whereas no significant differences were
observed between CE-MS peak signals using the three organic phases, this is not the case of the
background signal. ACN gave the highest background signal coming out the baseline at  1.5×107
arbitrary units, a.u., (depending on the ACN/water ratio and the sheath liquid flow), the MeOH gave a
baseline at  4×106 a.u. and then isopropanol at  1.5×106 a.u.. The best TIE and MS spectra were
obtained using a sheath liquid composed of 50:50 isopropanol:water (v:v) delivered at a flow of 0.29
mL/h, setting the dry gas temperature at 150C. The TIE and MS spectra of some selected peaks using
these optimized conditions can be observed in Figure 3. An increase by a factor close to 5 in the S/N
ratio was observed under these conditions (Figure 3) compared to the TIE obtained using 80% MeOH in
the sheath liquid (Figure 2), all other conditions being exactly the same. This is due, to a lower intensity
and less noisy baseline and to a slightly enhanced MS signal. However, looking at the MS spectra of
11
peak 9 in Figure 3, corresponding to the same MS spectra shown in Figure 2A-D (II), it can be seen that
the number of charges in each protein is now lower using isopropanol. In here, 3 main peaks are
observed in the spectra with a maximum at 2049.6 corresponding to 13 charges for a Mr of 26634,
whereas in Figure 2B (II), five clear ions were observed, with a maximum at 1776.8 corresponding to 15
charges for the same Mr. This was generally observed for all the peaks in the TIE, while some proteins
were only visible using 50% isopropanol. This is for example the case for peak 12, corresponding to a
protein of Mr = 27135. This might not only be due to the lower S/N ratio, but also to solubility
differences for certain proteins using the 80:20 (v:v) MeOH:water sheath liquid. For all other proteins, a
perfect match was obtained between the various Mr calculated using 80% MeOH and 50% isopropanol
in the sheath liquid. Comparing the MS spectra presented in Figure 3, it can be seen that their quality
varied a lot as can be deduced from the MS spectra for peaks 3 and 12. This could be due to protein comigration and, likely, formation of aggregates, either during the separation or in the spray chamber.
The precision in the determination of the Mr was also measured using 10 different experiments. An
average relative standard deviation (RSD(n=10)) lower than 0.02% was obtained.
3.3 CE-MS analysis of zein proteins from conventional and transgenic maize.
To corroborate the usefulness of the CE-MS method, zein proteins from three different maize cultivars
(Aristis, Tietar and PR33P66) were compared with their corresponding Bt genetically modified lines
(Aristis Bt, Tietar Bt and PR33P66 Bt, respectively). First, the six maize samples were studied by CEMS in order to characterize as many zein proteins as possible. Then, for each found protein, an extracted
ion electropherogram (EIE) was generated using all detected ions (i.e., m/z values) for this given protein.
For example, for a protein of Mr= 23270, m/z values of 1225.7; 1293.8; 1369.8; 1455.4; 1552.3; 1663.1;
1791.0; 1940.2 and 2116.4 (which correspond to 19+, 18+, 17+, 16+, 15+, 14+, 13+, 12+ and 11+ charges,
respectively) were used. In a second step, a protein will then be positively identified, if its EIE exhibits a
12
high intensity signal and an adequate electrophoretic profile (i.e., peak shape). Moreover, where
comigration was observed, only the most intense signals were used. More than 70 proteins were
distinguished in the first step, and used to generate their EIEs. However, less than 30 proteins were
selected in the second step. An example of analysis, using all the EIEs, for the Aristis cultivar can be
observed in Figure 4. As can be seen, this approach allows detecting multiple co-migrating peaks with
slightly different electrophoretic mobilities and relative molecular mass values. As can be seen in Figure
4, this is the case for example for peak 2 which is clearly composed of two main proteins, peak 4 is
composed of 4 main proteins, and peak 11 is composed of four main proteins. However, despite the
good separation achieved, this is still not sufficient in all cases. For instance, only 4 to 5 proteins can be
identified with enough accuracy and sensitivity in peaks 3, 5 and 6; however, the high level of noise of
the MS spectra (see for example the MS spectra of peak 3 in Figure 3), as well as the high degree of
convolution observed in Figure 4 suggest a much higher number of proteins. Peak 10 is shown to be
composed of a succession of proteins with very close Mr values (17156 to 17514). Another peak with
similar characteristic is observed with Tietar (peak labeled as 13 in Figure 4).
A comparison among the zein proteins profile obtained for the maize varieties Aristis, Aristis Bt, Tietar
and PR33P66 is shown in Figure 5. For each variety the UV trace, as well as the electrophoretic mobility
and Mr values of the main proteins are represented. The abscises have been transformed from migration
time to mobility using a two points calibration [23] using peaks 1 and 12 as reference (see Figure 4). As
an example, the comparison between Aristis and Aristis Bt maize (Figure 5A and 5B) demonstrates that
no detectable difference was observed between these varieties as corroborated by the data of Table 1.
The same was observed with the other lines when compared to their respective transgenic variety. This
is not surprising, since transgenic and its corresponding non-transgenic isogenic line cultivated under the
same conditions (as in the present case) should only differ by one protein, the Cry1Ab, corroborating
e.g., that no unexpected rearrangements of the new inserted gene able to generate some unexpected
protein have taken place. The fact that the Cry1Ab protein has not been detected is explained
13
considering that this protein is expressed at relatively high levels in the leaf (9 μg/g tissue) but at lower
concentration in the grain (<0.5 μg/g tissue)*. Besides, the extraction procedure has been optimized to
investigate the most complex protein fraction (i.e., zein fraction) and the Cry1Ab protein is expected to
come out in the globulin/albumin maize fraction.
Interestingly, some small differences were observed between the three conventional maize varieties
(Aristis, Tietar and PR33P66) as can be deduced from Figure 5 and Table 1. In this table, the relative
molecular mass and mobilities of the proteins common between two or more varieties are resumed.
Some results from literature are also given for comparison including data obtained from the work of
Woo et al. [24] (column 9), Wang et al. [6] (columns 10 and 11) and Adams et al. [7] (column 11).
Column 9 lists the predicted Mr of various zein proteins from the B73 inbred maize line based on
genomic analysis. Column 10 shows the approximated Mr values determined using SDS-PAGE, and
column 11 the measured Mr values using MALDI-TOF of the same line. Identification of the various
zein proteins observed by CE-MS (column 2) was done by molecular mass similarity with the B73 line
(column 8) whose proteins have been characterized by Woo et al. [24]. Correspondence among the
works by Woo et al. [24] (column 8) and Wang et al. [6] and Adams et al. [7] (column 10) were already
reported in these previous publications [6,7].
As can also be observed in Table 1, seven proteins of the nine described by Woo et al. [24] show a very
good match (less than 11 Da of difference) between the experimental values determined in the present
work by CE-MS (column 3) and the expected theoretical values based on genomic analysis
(corresponding to column B73 in Table 1). The fact that these proteins are, in most cases, present in the
Aristis, Tietar and PR33P66 varieties strongly suggests that these zein proteins are the same that the
ones determined in the B73 line. Moreover, as can be seen in Table 1, the protein Mr values determined
*
Data obtained from Monsanto web site (http://www.monsanto.com/monsanto/layout/)
14
by CE-MS are also in good agreement with the values determined using MALDI-TOF, and as expected
they are more accurate than the Mr values obtained by SDS-PAGE. Interestingly, the number of proteins
detected for the 19- and 22-kDa--zein family is higher using CE-MS (19 proteins detected by CE-MS
vs. 7 by MALDI-TOF [7]) what can be explained by the increased selectivity provided by the CE
separation. This, for example, allows us to clearly differentiate proteins with similar molecular mass the
19-22 kDa familly. This is the case for the proteins of Mr 24426, 24085, 24001, 24695, 24565 and
mobility 4.5, 5.6, 6.6, 7.3 and 8.4×10-9 m2 V-1 s-1, respectively. Such differentiation would have never
been possible by MALDI-TOF. Indeed, this complexity was first suggested by Adams et al. due to the
relatively broad peaks observed in MALDI-TOF, although, to our knowledge, this is the first
experimental demonstration of such effect. Besides, 15 kDa--zein and 16 kDa--zein were also
identified by CE-MS (entries 10 and 13 in Table 1), although these two entries correspond to the average
Mr of the numerous species as can be more clearly seen in peak 10 of Figure 4. In peak 10, six different
proteins of equivalent intensity were detected with an average Mr of 17294 and a standard deviation of
154, and in peak 13, four different proteins were detected with an average Mr of 17436 and a standard
deviation of 198. In spite of these good results, it is interesting to remark that the presence of 10-kDa -,
18-kDa - and 27-kDa -zein as described in several works [6,7,24] could not be corroborated by CEMS.
The most important variations between the three lines, as it can be observed in Figure 5, are due to the
peak migrating with a mobility of 5.6×10-9 m2V-1s-1. However due to comigration it is unclear whereas
this is due to variation in the concentration of the same proteins or to the presence of different proteins.
Differences in concentration can also be observed for the peak corresponding to the 15- (8×10-9 m2V-1s1)
and 16-kDa zein (9.5×10-9 m2V-1s-1). This result is consistent with what has previously been observed
by Adams et al. [7]. In their work, they also observed important variation in the 15- and 16-kDa zein
concentration between different varieties (in their case B73, M14, B37, Mo17, Oh43, W64A and W22).
Both the B37 and W64A were lacking the typical 15-kDa zein peaks. Variations can also be observed
15
between the group of peaks between 6.5 and 7×10-9 m2V-1s-1 that are due to variations in the abundance
of three 19 kDa--zein of Mr equal to 23361, 23352 and 24001, this is also in accordance with the work
of Adams et al. when comparing inbreds [7]. However, the CE-MS method proposed here allows
differentiating proteins with similar Mr values that have not been found in previous works. For example,
one of the differences between the cultivars are in the peaks at 7 and 7.5×10-9 m2V-1s-1 (entries 7 and 9
in Table 1, indicated by black arrows in Figure 5A, C and D). These two peaks correspond to two
proteins of similar Mr value, namely, 26753 and 26634, respectively. The total area of these two peaks
are constant and equal to 62.6×10-3 (±1.6%) a.u., however, in Aristis the main peak is at 7.5×10-9 m2V1s-1
(see Figure 5A), whereas with Tietar the main peak is at 7×10-9 m2V-1s-1 (see Figure 5C) while for
the PR33P66 variety, the peak at 7.5×10-9 m2V-1s-1 is inexistent (see Figure 5D).
Thus, these results demonstrate the great possibilities of the proposed CE-MS approach to get a more
complete picture about the zein protein fraction from maize. Besides, from our results it can tentatively
be concluded that the procedure is able to differentiate between varieties and to confirm that non
significant differences are observed between the conventional and transgenic isogenic lines as can be
deduced by comparing the columns Aristis and Aristis Bt in Table 1, or comparing their respective CEMS electropherograms given in Figure 5A and 5B. However, this point should be further confirmed
analyzing other families of proteins, metabolites, etc, for which other unintended effects could be
detected.
4. Concluding remarks
A method based on capillary electrophoresis-mass spectrometry has been developed for the analysis of
zein proteins. The method has been shown to be fast, reliable and provide accurate mass information.
This approach introduces a separation step based on proteins electrophoretic mobility prior to MS
16
allowing the differentiation of proteins of similar molecular mass what cannot be achieved by other
modern techniques including matrix-assisted laser desorption ionization time-of-flight mass
spectrometry. Zein proteins with small Mr differences (below 100 Da) were easily separated and
successfully analyzed by CE-MS. Thus, apart of the so-called 15-kDa--zein and 16-kDa--zein, which
are demonstrated to be formed by a heterogeneous group of proteins, numerous 19 and 22-kDa -zeins
were also identified for the first time in this work. The usefulness of this CE-MS method was
corroborated by comparing the Proteomic fingerprint of various maize lines including transgenic and
their corresponding non-transgenic isogenic lines cultivated under the same conditions. Some interesting
differences were observed between different lines of conventional maize (Aristis, Tietar and PR33P66).
On the other hand, no significant differences were observed in terms of zein content between nontransgenic and transgenic varieties what seems to corroborate the so-claimed substantial equivalence
between the studied varieties. However, this point needs to be confirmed further analyzing other
families of proteins, metabolites, etc.
ACKNOWLEDGMENTS
G.L.E. thanks the Spanish MEC for a postdoctoral grant. Authors are grateful to the AGL2005-05320C02-01 Project (Ministerio de Educacion y Ciencia) and the S-505/AGR-0153 ALIBIRD Project
(Comunidad Autonoma de Madrid) for financial support of this work.
17
5. References
[1]
Essen, A., Plant Physiol. 1986, 80, 623-627.
[2]
Essen, A., J. Cereal Sci. 1987, 5, 117-128.
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Monamy, F. A., Sessa, D. J., Lawton, J. W., Selling, G. W., J. Agric. Food Chem. 2006, 54, 543-
547.
[4]
Lawton, J. W., Cereal Chem. 2002, 79, 1-18.
[5]
Parris, N., Dickey, L.C., J. Agric. Food Chem. 2001, 49, 3757-3760.
[6]
Wang, J-F., Geil, P.H., Kolling, D.R.J, Padua, G.W., J. Agric. Food Chem. 2003, 51, 5849-5854.
[7]
Adams, W.R., Huang, A., Kriz, A.L., Luethy, M.H., J. Agric. Food Chem. 2004, 52, 1842-1849.
[8]
Rodriguez-Nogales, J.M., Garcia, M.C., Marina, M. L., J. Sep. Sci.2006, 29 ,197-210.
[9]
Issaq, H.J., Chan, K.C., Janini, G. M., Conrads, T. P., Veenstra, T. D., J. Chromatogr. B 2005,
817, 35-47.
[10]
Ishihama, Y., J. Chromatogr. A 2005, 1067, 73-83.
[11]
Stutz, H., Electrophoresis 2005, 26, 1254-1290.
[12]
Huck, C.W., Bakry, R., Huber, L.A., Bonn, G. K., Electrophoresis, 2006, 27, 2063-2074.
[13]
Simó, C., Barbas, C., Cifuentes A., Electrophoresis, 2005, 26 1306-1318.
[14]
Hernández-Borges, J., Neusüß, C., Cifuentes, A., Pelzing, M., Electrophoresis, 2004, 25, 2257-
2281.
[15]
Erny, G.L., Marina, M.L., Cifuentes, A., Electrophoresis (in press).
[16]
Simo, C., Elvira, C., Gonzalez, N., San Roman, J., Barbas, C., Cifuentes, A., Electrophoresis
2004, 25, 2056-2064.
[17]
Righetti, P.G., Gelfi, C., Verzola, B., Castelletti, L., Electrophoresis 2001, 22, 603-611.
[18]
Bean, S.R., Lookhart, G.L., Bietz, J.A., J. Agric. Food Chem. 2000, 48, 318-327.
[19]
Erny, G.L., Elvira, C., San Roman, J., Cifuentes, A., Electrophoresis 2006, 27, 1041-1049.
18
[20]
Nilsson, S.L., Bylund, D., Jorntén-Karlsson, M., Petersson, P., Markides, K. E., Electrophoresis
2004, 25, 2100-2107.
[21]
Geiser, L., Rudaz, S., Veuthey, J-L., Electrophoresis 2003, 24, 3049-3056.
[22]
Mirza, U. A., Cohen, S. L., Chait, B. T., Anal. Chem. 1993, 65, 1-6.
[23]
Erny, G. L., Cifuentes A., Electrophoresis 2007, 28, 1335–1344.
[24]
Woo, Y-M., Hu, D. W-N, Larkings, B.A., Jung, R., Plant Cell, 2001, 13, 2297-2317.
19
FIGURE CAPTIONS
Figure 1. CE-UV monitoring of zein protein extracted from Aristis maize (wild type) using as
extraction
solution:
A)
40:5:55
(v:v:v)
ACN:mercaptoethanol:water,
B)
60:5:35
(v:v:v)
ACN:mercaptoethanol:water, and C) 60:5:35 (v:v:v) ACN:mercaptoethanol:water, and adding
concentrated ammonium hydroxide to a final concentration of 120 mM. The separation was performed
in reverse polarity at -20 kV using a EPyM:DMA coated capillary with 37 cm of total length (30 cm of
detection length) and 50 μm i.d., thermostated at 25C. The BGE was composed of 40:20:2:38 (v:v:v:v)
ACN:isopropanol:formic acid:water. The detection was performed at 200 nm. The sample was injected
for 6 s at 3.5x103 Pa. See experimental for other conditions.
Figure 2. (I) Total ion electropherograms and (II) MS spectra corresponding to the peak marked with an
asterisk using different sheath liquids. The CE separation of zein proteins was performed in reverse
polarity (-20 kV) with an EPyM:DMA coated capillary of 80 cm length and 50 μm i.d.. The BGE was
composed of 60:2:38 (v:v:v) ACN:formic acid:water. The sample was injected for 45 s at 3.5x103 Pa. A)
Sheath liquid composed of 80:20 MeOH:H2O (v:v) delivered at 0.24 mL/h, dry gas: 250 C; B) Sheath
liquid composed of 90:10 MeOH:H2O (v:v) delivered at 0.10 mL/h, dry gas: 250 C; C) Sheath liquid
composed of 80:20 MeOH:H2O (v:v) delivered at 0.10 mL/h, dry gas: 250 C; D) Sheath liquid made of
80:20 MeOH:H2O (v:v) delivered at 0.24 mL/h, dry gas: 150 C. See experimental for other conditions.
Figure 3. Optimized CE-MS separation of zein proteins and some selected MS spectra. Separation
conditions: BGE: 40:20:2:38 (v:v:v:v) ACN:isopropanol:formic acid:water. Sheath liquid: 50:50
isopropanol:water (v:v) delivered at 0.29 mL/h. Dry gas at 150 C. Other conditions as in Figure 2.
20
Figure 4. Series of extracted ions electropherograms corresponding to the various proteins detected by
CE-MS in Figure 3.
Figure 5. Comparison of the zein proteins content of A) Aristis, B) Aristis Bt, C) Tietar and D)
PR33P66. Mr and mobility values were obtained from CE-MS separations. The cross correspond to the
relative molecular mass of the detected proteins. Migration times have been converted in electrophoretic
mobility values using a two points calibration. The electropherograms shown correspond to the UV
traces. All separation conditions as in Figure 3.
21
40
Intensity /10-3 a.u.
A
20
0
5
Time /min
7
8
6
Time /min
7
8
6
Time /min
7
8
B
40
Intensity /10-3 a.u.
6
20
0
5
C
Intensity /10-3 a.u.
40
20
0
5
Figure 1.
22
7
*
Count /107
3
2
Count /105
A(I)
2
1
A(II)
1776.8
1903.5
1412.9
1336.2
1
1665.7
2049.5
1567.6
0
0
16
22
20
Time /min
800
24
B(I)
3
Count /105
1
18
Count /107
*
B(II)
1776.8
1903.5
2049.5
1665.7
1
20
Time /min
22
800
24
C(I)
1200
m/z
C(II)
1530.3
1386.0
1
*
0
16
18
20
Time /min
800
D(I)
4
Count /105
2
1
0
16
18
20
Time /min
22
1792.2
1950.9
0
22
*
2000
1600
Count /105
Count /107
18
2
Count /107
2000
1600
0
16
3
m/z
2
0
4
1200
1200
m/z
2000
1600
D(II)
1665.71776.7
1567.6
2
1485.3
1903.5
2049.3
0
800
1200
1600
2000
m/z
Figure 2.
23
3
6
Cunts x107
4
4
1
2
5
9
8
7
2
10
11
12
0
30
32
2036.3
Peak 1
34
36
Time /min
1854.0
Peak 3
38
2008.6
42
Peak 5
1947.9
2184.8
1880.0
2124.7
1634.1 1745.9
1798.4
2049.6
1942.9
Peak 11
Peak 9
2092.2
Peak 12
1903.5
2088.1
1938.8
1776.6
1813.5
1593.7
1809.7
1700.0
1696.8
24
Figure 3.
6
3
5
9
11
12
8
4
1
7
10
2
30
13
34
38
42
Time /min
Figure 4.
25
B
A
25000
25000
Mr
Mr
20000
20000
15000
15000
4
5
6
7
9
2
8
-1
9
10
4
5
-1
7
8
2
-1
9
10
-1
Mobility /10 m V s
C
25000
6
9
Mobility /10 m V s
D
25000
Mr
Mr
20000
20000
15000
15000
4
5
6
7
9
2
8
-1
-1
Mobility /10 m V s
9
10
4
5
6
7
9
2
8
-1
Mobility /10 m V s
9
10
-1
Figure 5.
26
Table 1. Relative molecular mass and mobilities of zein proteins measured by CE-ESI-MS.
Peak
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
#11
#12
#13
Classical
Experimental
Mobility
Name
Mr by CE-MS
19 kDa  zein
19 kDa  zein
22 kDa  zein
22 kDa  zein
19 kDa  zein
19 kDa  zein
22 kDa  zein
22 kDa  zein
19 kDa  zein
19 kDa  zein
19 kDa  zein
22 kDa  zein
19 kDa  zein
19 kDa  zein
15 kDa  zein
22 kDa  zein
22 kDa  zein
22 kDa  zein
19 kDa  zein
19 kDa  zein
16 kDa  zein
23401 (±1)
24426 (±4)
26843 (±1)
26813 (±4)
24085 (±6)
23363 (±3)
26925 (±1)
26764 (±3)
23361 (±1)
23352 (±7)
24001 (±4)
26753 (±3)
24695 (±1)
26634 (±3)
17294 (see text)
27298 (±4)
27363 (±2)
27187 (±1)
24565 (±3)
27129 (±2)
17436 (see text)
/10-9 m2 V-1
s-1
4.5 (±0.2)
4.5 (ref)
5.0 (±0.1)
5.0 (±0.1)
5.6 (±0.1)
5.6 (±0.3)
5.9 (±0.1)
6.1 (±0.1)
6.4 (±0.1)
6.5 (±0.1)
6.6 (±0.1)
7.0 (±0.1)
7.3 (±0.1)
7.5 (±0.1)
8.0 (±0.1)
8.2 (±0.1)
8.2 (±0.1)
8.3 (±0.1)
8.4 (±0.1)
9.3 ((ref)
9.5 (±0.1)
a Data
obtained from reference [24].
b Data
obtained from reference [6].
Aristis
Y
Aristis
Bt
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Tietar
PR33P66
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
B73a
SDSPAGEb
MALDI-TOFMS
24000
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
24087
23359
26923
22000
24069c, 24087b
23318c
26308c
23359
22000
23318c, 23362b
26751
24706
26741c
24644c
17458
17125c
27128
17663
17714c
27
c Data
obtained from reference [7].
28