Research Article
Integration of capillary isoelectric focusingwith monolithic immobilized pH gradient,immobilized trypsin microreactor andcapillary zone electrophoresis for on-lineprotein analysis
An integrated platform consisting of protein separation by CIEF with monolithic
immobilized pH gradient (M-IPG), on-line digestion by trypsin-based immobilized
enzyme microreactor (trypsin-IMER), and peptide separation by CZE was established. In
such a platform, a tee unit was used not only to connect M-IPG CIEF column and trypsin-
IMER, but also to supply adjustment buffer to improve the compatibility of protein
separation and digestion. Another interface was made by a Teflon tube with a nick to
couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube
filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric
contact for peptide separation. By such a platform, under the optimal conditions, a
mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M-IPG
CIEF, followed by on-line digestion by trypsin-IMER and peptide separation by CZE.
Many peaks of tryptic peptides, corresponding to different proteins, were observed with
high UV signals, indicating the excellent performance of such an integrated system. We
hope that the CE-based on-line platform developed herein would provide another powerful
alternative for an integrated analysis of proteins.
Keywords: Capillary zone electrophoresis / Immobilized enzyme microreactor /Integrated platform / Monolithic immobilized pH gradient / TrypsinDOI 10.1002/jssc.201000324
1 Introduction
Top-down and bottom-up approaches are two complemen-
tary strategies developed for MS-based protein identification
and characterization [1–4]. For top-down strategy, usually
after prefractionation, intact proteins are directly analyzed
by MS. Although the identification accuracy might be high
without cleavage of proteins into peptides, the obtained
protein information might be less, limited by MS/MS
identification capacity. For bottom-up strategy, proteins are
generally digested into peptides before separation by HPLC
and identification by MS, which could offer high resolution
and high throughput for the analysis of complex protein
samples. However, the simultaneous separation of large
number of peptides does not only exert tremendous
challenge to separation columns, but also may result in
false-positive protein identification results by MS/MS [2].
Therefore, protein separation before digestion might be a
good solution to combine the advantages of both top-down
and bottom-up strategies.
As one important separation method, CE has drawn
much attention especially for the separation of large
biomolecules, such as proteins [5, 6]. Among various CE-
based separation modes, CIEF offers high resolution
and high sensitivity. However, in traditional CIEF, the
addition of ampholytes in buffer is indispensable to estab-
lish a stable pH gradient, which may not only decrease UV
detection sensitivity at low wavelengths, but also prevent the
direct hyphenation of CIEF with MS. To solve this problem,
either low concentration ampholytes were used [7], or
ampholytes were removed before entering MS [8, 9].
Another promising approach is to immobilize ampholytes
onto supporting materials (i.e. monolithic matrix) to form a
stable pH gradient [10, 11], avoiding the additional steps
for ampholyte removal and possibly expanded diffusion of
focused zones.
Tingting Wang1,2
Junfeng Ma1
Guijie Zhu1
Yichu Shan1
Zhen Liang1
Lihua Zhang1
Yukui Zhang1
1Key Laboratory of SeparationScience for AnalyticalChemistry, NationalChromatographic Research andAnalysis Center, Dalian Instituteof Chemical Physics, ChineseAcademy of Science, Dalian,P. R. China
2Graduate University of ChineseAcademy of Sciences, Beijing,P. R. China
Received May 8, 2010Revised July 27, 2010Accepted July 27, 2010
Abbreviations: IMER, immobilized enzyme microreactor;LPAA, linear polyacrylamide; c-MAPS, 3-methacryloxy-propyltrimethoxysiliane; M-IPG CIEF, capillary isoelectricfocusing with monolithic immobilized pH gradient
Correspondence: Professor Lihua Zhang, Key Laboratory ofSeparation Science for Analytical Chemistry, National Chroma-tographic Research and Analysis Center, Dalian Institute ofChemical Physics, Chinese Academy of Science, 457 ZhongshanRoad, Dalian 116023, P. R. ChinaE-mail: [email protected]:186-411-84379779
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2010, 33, 3194–32003194
To achieve high-throughput proteome analysis, after
protein prefractionation, on-line protein digestion is indis-
pensable, which can be achieved by immobilized enzyme
microreactors (IMERs), with enzymes (typically trypsin)
covalently bonded, trapped or physically adsorbed onto
particles, membranes, the inner walls of fused-silica capil-
laries or monolithic materials [12–15]. In contrast to the
traditional in-solution digestion, IMERs exhibit higher
enzymatic activity and faster digestion speed, and more
importantly, IMERs can be easily coupled with separation
systems for on-line protein analysis [12, 15].
To date, many efforts have been made to combine on-
line protein digestion and protein and/or peptide separa-
tion. Amankwa and Kuhr [16] combined trypsin-modified
capillary microreactor to the capillary for CZE separation viaa 100-mm-solution gap. Kato et al. [17] integrated on-line
protein digestion, peptide separation and protein identifi-
cation by ESI-MS. The photopolymerized sol–gel monolith
immobilized with pepsin was initially fabricated at the inlet
of the capillary. The resulting mixture of peptides could be
directly separated in the portion of the capillary where no
photopolymerized sol–gel monolith existed. Recently,
Schoenherr et al. [18] presented a proof-of-concept system of
CZE-IMER-CZE-ESI-MS/MS for on-line protein separation,
digestion, peptide separation and identification. In their
system, to be compatible with the low pH values for protein
and peptide separation, a pepsin-based IMER, which works
under acidic conditions, was fabricated at the distal end of a
protein separation capillary and connected to a peptide
separation capillary via a finely machined interface.
In this paper, a novel integrated CE-based platform was
established, consisting of M-IPG CIEF (capillary isoelectric
focusing with monolithic immobilized pH gradient) for
protein separation, trypsin-IMER for on-line digestion and
CZE for peptide separation, with a tee unit and a Teflon
tube-based interface to couple different units, improve the
buffer compatibility and keep electric contact of the whole
separation system. With such a platform, a 3-protein
mixture was successfully analyzed with high efficiency, high
accuracy and high throughput.
2 Materials and methods
2.1 Apparatus
All CE experiments were performed on TriSep-2010GV,
equipped with a Data Module UV-visible detector and a
high-voltage power supply (Unimicro Technologies, Plea-
santon, CA, USA). A Workstation Echrom 98 of Dalian Elite
Analytical Instrument (Dalian, P. R. China) was used for
data acquisition. Syringe pump was purchased from
Baoding Longer Precision Pump (Baoding, P. R. China).
All the proteins were desalted by Shimadzu HPLC system
equipped with a UV-absorbance detector (Tokyo, Japan),
and the eluants were lyophilized in a SpeedVac (Thermo
Fisher, San Jose, CA, USA).
2.2 Reagents and materials
N,N,N0N0-Tetramethylethylenediamine (TEMED) (98%),
ammonium persulfate (98%), acrylamide, tetraethoxysilane
(95%), 3-aminopropyltriethoxysilane (97%), and N,N0-methyl-
enebisacrylamide were purchased from Acros Organics
(Geel, Belgium). Glycidyl methacrylate and trypsin inhibitor
from lima bean (pI 4.5) were ordered from Fluka (St.
Gallen, Switzerland). Ampholine (pH 3.5–10.0), trypsin
from bovine pancreas, myoglobin from equine skeletal
muscle (pI 7.3), carbonic anhydrase from bovine erythro-
cytes (pI 6.6), bovine insulin (pI 5.3), 3-methacryl-
oxypropyltrimethoxysiliane (g-MAPS, 98%), and b-lacto-
globulin from bovine milk (pI 5.2) were bought from Sigma
(Steinheim, Germany). Azobisisobutyronitrile was obtained
from The Fourth Shanghai Regent Plant (Shanghai, P. R.
China). PEG (MW 8000, 10 000) and BSA (pI 4.9) were from
Sino-American Biotechnology Company (Shanghai, P. R.
China). Ribonuclease A (pI 9.5) was purchased from Merck
(Darmstadt, Germany). Dithioerythritol and iodoacetic acid
were obtained from Amresco (Solon, OH, USA). Water was
purified by a Milli-Q system (Millipore, Molsheim, France).
All inorganic reagents were of analytical reagent grade.
Fused-silica capillaries (25-mm id� 375-mm od, 50-mm
id� 375-mm od, 100-mm id� 375-mm od) were bought from
Kailiaola Chromatographic Analysis (Handan, P. R. China).
Teflon tube (1/1600) was from J & J Industries (Beijing,
P. R. China).
2.3 Preparation of M-IPG column
Fused-silica capillary was treated successively by 0.5 mol/L
HCl, water, 0.5 mol/L NaOH, water and methanol for 30 min,
respectively, and dried with N2 at 701C for 1 h. Then
the capillary was filled with a solution of g-MAPS (50% v/v
in methanol), and kept at room temperature with both
ends sealed for 24 h. Unreacted g-MAPS was washed
with methanol and the capillary was purged with N2 at 701C
for 1 h.
The procedure of M-IPG column preparation was as
follows. In brief, 20.6 mg glycidyl methacrylate and 34.3 mg
ampholines were dissolved in 679.4 mg formamide. After
vortexing for a few minutes, the mixture was put in a water
bath at 401C for 1 h, and then at 41C for 10 min to slow
down the reaction between amine and epoxy groups.
Subsequently, 10.0 mg acrylamide, 20.0 mg N,N0-methyl-
enebisacrylamide, 45.0 mg PEG-8000 and 25.0 mg PEG-
10 000 were added into the mixture. After the addition of
azobisisobutyronitrile (1 wt% respect to monomers), the
solution was mixed completely, and degassed with N2 for
5 min. Then the polymerization solution was injected into a
g-MAPS-coated capillary (100-mm id� 375-mm od), followed
by focusing at 400 V/cm, with 20 mmol/L glutamic acid as
the anolyte buffer, and 20 mmol/L NaOH as the catholyte
buffer. After 6 min, with both ends sealed, the capillary
was put at 701C for 20 h, to immobilize the pH gradient on
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the monolith. Finally, the prepared M-IPG column was
flushed with ethanol for 2 h, followed by water for 1 h.
2.4 Preparation of trypsin-IMER
A trypsin-IMER (100-mm id� 375-mm od) was prepared by
the immobilization of trypsin on hybrid silica monolith
according to our previous work [19]. Briefly, the monolithic
matrix was formed with tetraethoxysilane and 3-amino-
propyltriethoxysilane as two precursors under the presence
of cetyltrimethyl ammonium bromide. Then, the support
was activated with glutaraldehyde, followed by trypsin
immobilization via covalent bonding. In the following
experiments, unless specified otherwise, the IMER length
was 1 cm and protein digestion was carried out at room
temperature.
2.5 Coating of CZE capillary
The capillary (50-mm id� 375-mm od or 25-mm id� 375-mm
od) was coated with linear polyacrylamide (LPAA) by a
similar procedure as described by Schmalzing et al. [20]. In
brief, a solution of 25.0 mg acrylamide in 0.5 mL water was
degassed for 30 min by N2. Aliquots of TEMED (8 mL) (10%,
v/v in water) and ammonium persulfate (8 mL) (10%, w/v in
water) were then added into the solution, and the mixture
was immediately injected into g-MAPS-coated capillary.
After polymerization for 12 h, the capillary was rinsed with
water to wash off excessive polyacrylamide.
2.6 Sample preparation
Following the previous protocol [19], protein (1 mg) was
dissolved in 100 mL 8 mol/L urea and then reduced in
10 mmol/L dithiothreitol for 1 h at 561C. When cooled to
room temperature, cysteines were alkylated in the dark in
20 mmol/L iodoacetic acid for 30 min at room temperature,
followed by dilution with 1 mL 50 mmol/L Tris-HCl (pH
8.0) to decrease the urea concentration. Protein was desalted
by a home-made C8 trap column, and then the eluant was
lyophilized and redissolved in 10 mmol/L Tris-HCl (pH 8.0)
to the desired concentration.
2.7 M-IPG CIEF separation
A 29-cm-long M-IPG column (with the effective length
of 21 cm) was used for M-IPG CIEF separation, with
20 mmol/L NaOH and 20 mmol/L glutamic acid as the
cathode and anode buffer, respectively. The sample was
injected to fill the whole capillary. Then a voltage of 10 kV
was applied for focusing. The sample zones were pushed
through the detection window by a manual pump, and
detected at 214 nm with a UV detector.
2.8 IMER-CZE analysis
A syringe pump was used to push 1 mg/mL myoglobin (in
10 mmol/L Tris-HCl, pH 8.0) through the trypsin-IMER at
various flow rates. The outlet end of trypsin-IMER was
coupled with CZE by Teflon tube via a short capillary (with a
length of 3 cm, 25-mm id� 375-mm od), which was
immerged in 20 mmol/L glutamic acid (pH 3.4).
The LPAA-coated capillary (with an effective length of
20 cm, 50-mm id� 375-mm od) was used for the CZE
separation. The injection volume was optimized and
peptides were detected by UV at 214 nm.
3 Results and discussion
3.1 Setup of M-IPG CIEF-IMER-CZE platform
The schematic diagram of integrated CE-based platform is
shown in Fig. 1. To improve the buffer compatibility in the
whole platform, M-IPG CIEF for protein separation was
performed off-line, and then coupled to trypsin-IMER via a
tee unit. The focused sample zones were mobilized into
IMER via syringe pump 1, and simultaneously syringe
pump 2 was triggered to apply the adjustment buffer to
improve the biocompatibility of protein buffer and on-line
tryptic digestion. With an optimal flow rate of the sample
Figure 1. Schematic diagram of M-IPG CIEF-IMER-CZE platform. (1) and (10) capillaries;(2) and (20) Teflon tubes; (3) centrifuge tube;(4) hole for buffer adding; (5) hole forplatinum wire; (6) nick on Teflon tube; (7)gap between capillaries.
J. Sep. Sci. 2010, 33, 3194–32003196 T. Wang et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
and adjustment buffer, as well as mobilization time interval,
protein digests were further separated by CZE.
3.2 Performance evaluation of M-IPG CIEF
As shown in Fig. 2A, for a 4-protein mixture consisting of
myoglobin (pI 7.3), carbonic anhydrase (pI 6.6), insulin
(pI 5.3) and trypsin inhibitor (pI 4.5), baseline separation
was achieved with M-IPG CIEF, demonstrating the good
resolution of M-IPG column. Although myoglobin and
carbonic anhydrase only yielded roughly baseline separa-
tion, the resolution between them was acceptable (1.5),
demonstrating that proteins with even 0.7 pH unit
difference could be well resolved. In addition, the relation-
ship between pI and the migration time of four proteins was
studied. The migration time herein was set as the time used
to mobilize the samples from their focused position to the
detection window. From Fig. 2B, it could be seen that good
linearity was obtained (R2 5 0.9869), implying that the
separation mechanism was the same as the traditional
free-solution CIEF.
3.3 Hyphenation of M-IPG CIEF with trypsin-IMER
via a tee unit
Although the hybrid silica monolith-based trypsin-IMER
had demonstrated excellent digestion efficiency even at
room temperature [19, 21], buffer exchanging was adopted
herein since the pH value of mobilized sample zones from
M-IPG CIEF ranged from pH 3.5 to 10.0, which was not
compatible with tryptic digestion (with an optimal pH
�8.0). To this end, a tee unit was used to adjust the pH
value of protein fractions from M-IPG CIEF before
pumping through the trypsin-IMER.
To obtain compatible buffer conditions, syringe pumps
1 and 2 were connected to the tee unit to introduce
sample and adjustment buffer, respectively, and the pH of
eluted mixture was measured by a piece of pH test paper.
To mimic the extreme pH values for M-IPG CIEF,
20 mmol/L glutamic acid (pH 3.4) and lysine (pH 9.9) were
respectively introduced by syringe pump 1 with different
flow rates, while five kinds of adjustment buffers, including
50 mmol/L Tris-HCl, 25 mmol/L Tris-HCl, 50 mmol/L
Na2HPO4, 50 mmol/L Tris-H3BO3, and 50 mmol/L Tris-
H3PO4 (pH 8.0), were respectively introduced with a
constant flow rate by syringe pump 2 (1 mL/min). Although
both 50 mmol/L Na2HPO4 and 50 mmol/L Tris-H3BO3
(pH 8.0) illustrated appropriate buffer exchanging capacity
(Table 1), 50 mmol/L Na2HPO4 (pH 8.0) was chosen as the
optimal adjusting buffer due to the possible over-dilution of
samples and the UV absorption disturbance resulting from
50 mmol/L Tris-H3BO3. With an optimal flow rate ratio of
sample and adjustment buffer as 1:1, the pH of eluted
buffer was efficiently adjusted to near pH 8.0, compatible
with the downstream tryptic digestion.
For the coupling system, with b-lactoglobulin
(0.1 mg/mL) as the sample, flow rates of the sample and
adjustment buffer were examined. The results showed that
the protein buffer was neutralized to pH �8.0 when mobi-
lizing the focused protein at a flow rate of 290 nL/min,
further demonstrating that 50 mmol/L Na2HPO4 (pH 8.0)
and the flow rate ratio of sample and adjustment buffer as
1:1 could be used for the on-line system.
3.4 Hyphenation of trypsin-IMER with CZE by Teflon
tube interface
Since the optimal CZE buffer for peptide separation
(20 mmol/L glutamic acid, pH 3.4) was quite different from
the pH value of tryptic digests eluted from trypsin-IMER
(pH 8.0). Another interface, a Teflon tube interface, was
adopted to acidify tryptic digests and serve as the running
buffer for CZE separation. As shown in Fig. 1, a short
capillary (with a length of 3 cm, 25-mm id� 375-mm od) was
Figure 2. Separation of a mixture of standard proteins by M-IPGCIEF (A) and the linearity of pI versus migration time (B).Experimental conditions: M-IPG column, 29 cm total length,21 cm effective length, 100-mm id � 375-mm od; catholyte,20 mmol/L NaOH; anolyte, 20 mmol/L glutamic acid; voltage,345 V/cm; detection wavelength, 214 nm; sample, myoglobin(pI 7.3), carbonic anhydrase (pI 6.6), insulin (pI 5.3) and trypsininhibitor (pI 4.5), dissolved in 10 mmol/L Tris-HCl buffer (pH 8.0),0.1 mg/mL of each protein.
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used to connect trypsin-IMER and CZE capillary (10) by a
2.0-cm-long Teflon tube (20), with a nick on the surface
(10–20 mm) (6). Capillaries (1) and (10) were cut smoothly,
and the gap (7) between them was 10–20 mm. Both the nick
and the gap were designed to exchange protein digests
buffer and keep electric contact for CZE-based peptide
separation. The capillaries were further fixed into a
centrifuge tube (3) filled with CZE buffer via two small
pieces of Teflon tubes (2). Two holes were made on the
centrifuge tube, one (4) for buffer addition, and the other (5)
for inserting platinum wire. During CZE separation, syringe
pumps 1, 2 and the Teflon tube interface were grounded.
Compared to other reported vial, valve or tee-piece interfaces
[22], not only the interface was simplified, but also the brand
broadening was avoided.
Under the optimal conditions, the RSD (%) of the
migration time of ribonuclease A in 21 consecutive runs was
about 2.3%, and no leakage or bleeding of the coating
material was observed in 24 h, demonstrating the good
stability of our prepared LPAA coating.
Table 1. Comparison of pH adjustment capacity of different buffers
Syringe pump 1 Syringe pump 2 (1 mL/min, pH 8.0)
Buffer Flow rate
ratioa)
50 mmol/L
Tris-HCl
25 mmol/L
Tris-HCl
50 mmol/L
Na2HPO4
50 mmol/L
Tris-H3BO3
50 mmol/L
Tris-H3PO4
20 mmol/L Glutamic acid (pH 3.4) 0.25 8.0b) 8.0 8.0 8.0 8.0
0.50 8.0 7.0 8.0 8.0 8.0
0.75 7.5 6.5 8.0 8.0 7.5
1.00 7.0 5.0 8.0 8.0 7.0
2.00 5.0 4.0 7.0 7.0 6.0
20 mmol/L Lysine (pH 9.9) 0.25 8.0 8.0 8.0 8.0 8.0
0.50 8.0 8.0 8.0 8.0 8.0
0.75 8.0 8.0 8.0 8.0 8.0
1.00 8.0 8.0 8.0 8.0 8.0
2.00 9.0 9.0 9.0 8.5 8.0
a) Flow rate ratio: the flow rate of syringe pump 1/the flow rate of syringe pump 2.
b) pH values determined by the pH test paper.
Figure 3. Effect of injection time of 50 mmol/L Na2HPO4 (pH 8.0)buffer on CZE current in the IMER-CZE unit. Experimentalconditions: sample, 50 mmol/L Na2HPO4 (pH 8.0); the flow rateof syringe pump, 150 nL/min; CZE capillary coated with LPAA,50-mm id�375-mm od, 30 cm total length, 20 cm effective length;electric field strength, 266 V/cm; separation buffer: 20 mmol/Lglutamic acid.
Figure 4. Effect of injection time (A) and flow rate (B) on theseparation of myoglobin digests for the IMER-CZE unit. Experi-mental conditions: capillary coated with LPAA, 50-mm id� 375-mm od, 30 cm total length, 20 cm effective length; detectionwavelength, 214 nm; separation buffer, 20 mmol/L glutamic acid;IMER, 1 cm length, 100-mm id�375-mm od; sample, myoglobin,dissolved in 10 mmol/L Tris-HCl buffer (pH 8.0), 0.1 mg/mL;electric field strength, 266 V/cm; for (A), the flow rate of syringepump, 270 nL/min; for (B), the time of syringe pump, 0.3 min.
J. Sep. Sci. 2010, 33, 3194–32003198 T. Wang et al.
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To evaluate the buffer exchange capability of such a
simple interface, 50 mmol/L Na2HPO4 (pH 8.0) was
pumped at a flow rate of 150 nL/min for different time
periods. As shown in Fig. 3, with the injection time
increased, the CZE current was first increased, and then
decreased to current without injection (2.3 mA). The longer
the injection time, the longer the time needed for sample
buffer to be completely diluted by CZE buffer. In addition, if
the injection time was over 0.6 min, the current could be
hardly resumed to 2.3 mA. Therefore, the injection time of
samples (e.g. 50 mmol/L Na2HPO4, pH 8.0) should be less
than 0.6 min at the flow rate of 150 nL/min, i.e. the injection
volume should be less than �90 nL.
With the digests of 1 mg/mL myoglobin from the
trypsin-IMER as a sample, the CZE injection volume was
further investigated with either fixed injection flow rate or
time. As shown in Fig. 4A and B, excellent separation was
achieved with an optimal introduction flow rate of 270 nL/
min and an injection time of 0.3 min. It is noteworthy that
the total injection volume was �80 nL, within the range
mentioned above (o90 nL).
3.5 Application of M-IPG CIEF-IMER-CZE
With the above-mentioned optimal conditions, a mixture of
ribonuclease A, myoglobin and BSA (0.10 mg/mL each) was
analyzed by the integrated platform. After focusing with
M-IPG CIEF, the proteins were continuously transferred
to the trypsin-IMER by syringe pump 1 at the flow rate of
78 nL/min for 0.5 min, and simultaneously adjusted by
50 mmol/L Na2HPO4 (pH 8.0) via syringe pump 2 with the
flow rate of 81 nL/min for 0.5 min. The corresponding
volume of injected peptides for CZE was �80 nL and the
residence time of proteins in the trypsin-IMER was �28 s.
As shown in Fig. 5, proteins in 12 fractions eluted from M-
Figure 5. Analysis of a mixtureof ribonuclease A, myoglobinand BSA by M-IPG CIEF-IMER-CZE. Number ‘‘1’’, ‘‘2’’,y and‘‘12’’ represented the sequen-tially eluted fractions from M-IPG CIEF and trypsin-IMERfollowed by separation withCZE. Experimental conditions:for the CZE separation: coatedwith LPAA, 50-mm id� 375-mmod, 30 cm total length, 20 cmeffective length; electric fieldstrength, 266 V/cm; detectionwavelength, 214 nm; separa-tion buffer, 20 mmol/L gluta-mic acid; for the trypsin-IMER:IMER, 1 cm length, 100-mm id �375-mm od; for the M-IPG CIEFseparation: M-IPG column,20 cm length, 100-mm id � 375-mm od; anolyte, 20 mmol/Lglutamic acid; catholyte,20 mmol/L NaOH; electric fieldstrength for focusing, 345 V/cm; for the system: buffer insyringe pump 1, 5 mmol/L Tris-HCl pH 8.0; buffer in syringepump 2, 50 mmol/L Na2HPO4
pH 8.0; the flow rate of syringepump 1, 78 nL/min, the flowrate of syringe pump 2, 81 nL/min; the injected time of everyfraction, 0.5 min; sample, ribo-nuclease A (pI 9.5), myoglobin(pI 7.3) and BSA (pI 4.9),dissolved in 10 mmol/L Tris-HCl buffer (pH 8.0), 0.1 mg/mLof each protein.
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IPG CIEF were further on-line digested by trypsin-IMER,
and separated by CZE. The on-line digests were separated
preferably and resolved many peaks, indicating the excellent
performance of such an integrated system. According to the
peptides profiling and each protein concentration in the
original sample, fractions 1–3 might be the digests of BSA, 5
and 6 might be from myoglobin, 9 and 10 might be from
ribonuclease A. Fractions 4, 7 and 8 might be peptides from
the mixture of neighbored proteins. It should be noted that
almost all samples were successfully transferred and
analyzed, as could be seen from the few signals recorded
from fractions 11 and 12. In addition, the large peak shown
at the beginning of each figure might be induced by buffer
exchange. Although the integrated M-IPG CIEF-IMER-CZE
platform exhibited preliminary success for on-line protein
analysis, further automation and protein identification need
to be performed to achieve higher analytical throughput and
higher sensitive identification. The related work concerning
automated operation units and the coupling of M-IPG CIEF-
IMER-CZE system with ESI-MS/MS for on-line protein
analysis is being carried out in our lab.
4 Concluding remarks
An integrated CE-based platform involving protein
separation by M-IPG CIEF, on-line digestion by
trypsin-IMER and peptide separation by CZE was developed
for protein analysis. To improve the compatibility of
different units, two interfaces were applied between M-
IPG CIEF and trypsin-IMER, as well as between trypsin-
IMER and CZE. Under optimal conditions, a 3-protein
mixture was successfully separated, demonstrating the
potential of such an integrated platform for the analysis of
complex samples.
Further efforts to improve the automation and detection
sensitivity of such an integrated platform, including on-line
focusing and automatic migration of samples by M-IPG
CIEF and coupling the M-IPG CIEF-IMER-CZE system with
ESI-MS/MS, is being carried out for high-throughput
protein analysis.
The authors are grateful for the financial support fromNational Basic Research Program of China (2007CB914100),National Natural Science Foundation (20935004), KnowledgeInnovation Program of Chinese Academy of Sciences(KJCX2YW.H09) and Sino-German Cooperation Project(GZ 3164).
The authors have declared no conflict of interest.
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