Integration of capillary isoelectric focusing with monolithic immobilized pH gradient, immobilized...

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

J. Sep. Sci. 2010, 33, 3194–3200 Electrodriven Separations 3195


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.


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.



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.



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.



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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Integration of capillary isoelectric focusing with monolithic immobilized pH gradient, immobilized...

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