J. Mass Spectrom. Soc. Jpn.

Vol. 50, No. 1, 2002

COMMUNICATION

Capillary Isoelectric Focusing Separation Combined with Mass
Spectrometry Using Sonic Spray Ionization for Protein Analysis
Yukiko H>G676N6H=>῎a) and Atsumu H>G676N6H=>a)(Received September 19, 2001; Accepted October 26, 2001)
Capillary isoelectric focusing (CIEF) separation combined with mass spectrometry for protein analysis has
been demonstrated by using a sonic spray ionization (SSI) interface, which has a bu#er reservoir between the ion
source and the separation capillary. Since the bu#er reservoir was used as an outlet reservoir for CIEF, on-line
and one-step CIEF/MS was possible. In SSI, since a sample solution is sprayed at any solution-ﬂow rate from asample-introduction capillary with a high-speed gas ﬂow, high-polymer ampholytes for CIEF can be usedwithout clogging up the spray nozzle of the interface. Also, additives such as ampholytes signiﬁcantly decreaseion intensity of analyte molecules; however, the SSI interface whose reservoir is ﬁlled with an acetic acid solution
minimizes the decrease in ion intensity of the analyte. We combined CIEF with an ion trap mass spectrometer
and were able to detect 160 fmol of myoglobin and cytochrome-c.

Introduction
Separation and identiﬁcation of proteins and peptides from cell-extracts are becoming important in theﬁeld of proteomics. The isoelectric point (pI) of proteins is a useful parameter for identifying proteins aswell as their molecular weights. For this identiﬁcation,
isoelectric focusing separation in a capillary,1) i.e., capillary isoelectric focusing (CIEF), is a powerful methodfor separation of proteins according to pI. Coupling
CIEF and mass spectrometry2)῍9) are available for fasterprotein characterization and higher reproducibility
than two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In these techniques, sheath-ﬂow electrospray ionization (ESI) is used as an interface. However, they are not simple procedures; that is, they involve either of two types: after CIEF separation between the inlet and outlet reservoirs, one end of theseparation capillary is removed from the reservoir andinserted into a sheath-ﬂow interface3), 5), 7)῍9); or theoutlet catholyte is exchanged immediately for an
acidic sheath liquid in the sheath-ﬂow interface.4)
We have recently developed a sonic spray ionization(SSI) interface for capillary electrophoresis/mass spectrometry (CE/MS): it has a bu#er reservoir between thesample-introduction capillary of the ion source and theelectrophoresis-separation capillary.10) In SSI, a sample
solution is sprayed from a sample-introduction capillary by a high-speed nitrogen-gas ﬂow that is coaxial tothe capillary, and ions of the chemicals in the solution,
as well as charged droplets, are produced at room
temperature and atmospheric pressure.11)῍14) Since thesolution is sprayed only by the gas ﬂow, it can be usedwith a wide range of bu#er solutions and solution-ﬂowrates,10), 14) and high-polymer ampholytes for CIEF canbe used without clogging up the spray nozzle of the῎a) Central Research Laboratory, Hitachi, Ltd. (1῍280 Higashikoigakubo, Kokubunji, Tokyo 185῍8601, Japan)
Tel: ῌ81῍42῍323῍1111, Fax: ῌ81῍42῍327῍7681
E-mail: hiraby@crl.hitachi.co.jp

interface. In the SSI interface for CE/MS, an aceticacid solution in the bu#er reservoir is pumped into thesample-introduction capillary by the pressure reduction around the tip of this capillary due to the highspeed gas ﬂow; thus, the mobile-phase bu#er, containing nonvolatile compounds such as phosphate in thesample-introduction capillary, is mixed with the acetic
acid.10) This mixing increases the ion intensity of thesample 100-fold by enhancing the evaporation ofcharged droplets.10) Furthermore, since the bu#er reservoir is also used as the outlet reservoir, one-stepCIEF/MS analysis is possible. Using this SSI technique, we performed CIEF/MS analysis of a protein
mixture.
Experimental
Materials
Carbonic anhydrase II (CA II, pI 5.4 and 5.9) andmyoglobin (Mb, pI 6.8 and 7.2) for pI makers werepurchased from Sigma. Cytochrome-c (Cyt-c, pI 10.1)
was also purchased from Sigma. Pharmalyte (pI 3-10)
was purchased from Amersham Pharmacia Biotech.
CIEF/SSI-MS system
Figure 1 is a cross-sectional view of the SSI interface.
The details are described elsewhere.10) The sonic sprayion source, which is a part of a three-dimensional quadrupole (3DQ) ion-trap mass spectrometer (M-8000, Hitachi, Ltd., Tokyo), is modiﬁed. Namely, one end of afused-silica sample-introduction capillary (200-mm o.d.,
100-mm i.d., 10 cm long) is inserted into an oriﬁce (0.4mm i.d.) in the housing of the ion source. Pressurized
nitrogen gas ﬂows into the housing, causing a gas ﬂow
through the oriﬁce to the atmosphere, which thereby
generates a spray. A voltage of ῍1.2 kV is applied tothe housing, which is isolated from the solution by thesample-introduction capillary.10) The other end of thesample-introduction capillary is inserted into a Teﬂontube (0.25-mm i.d., 1.7-mm o.d.) that passes through abu#er reservoir. The bu#er reservoir is ﬁlled with ananolyte, so that the pinhole in the Teﬂon tube is submerged and the acetic acid solution ﬂows through theῌ 21 ῌ

Y. Hirabayashi and A. Hirabayashi

Fig. 1.

Cross-sectional view of the sonic spray interface
and CIEF-SSI/MS system.

pinhole into the sample-introduction capillary becauseof pumping e#ect.10) The electrical potential of thesolution in the sample-introduction capillary and abu#er reservoir is held at ground through the electrodeof the bu#er reservoir. A fused-silica capillary forseparation (i.e., the separation capillary: 50-mm i.d., 375mm o.d., 80 cm long) taken from a CAPI-3200 electrophoresis system (Otsuka Electronics Co., Ltd., Osaka) isinserted into the Teﬂon tube from the opposite end.
The capillaries are set opposite each other near thepinhole in the tube. The other end of the separation
capillary goes into a mobile-phase reservoir ﬁlled withthe catholyte in CAPI-3200. A voltage is applied to thecatholyte in the mobile-phase reservoir to generate a
pH gradient for isoelectric focusing. After focusing,
the samples pushed out from the separation capillary
are mixed with the acetic acid solution in the area
beneath the pinhole in the tube, and pumped into thesample-introduction capillary. The sample solution isthen sprayed from the sample-introduction capillaryby the sonic gas ﬂow. The ions produced in the atmosphere are introduced into the vacuum region of theM-8000 mass spectrometer through a sampling oriﬁce.
CIEF separation
The separation capillary coated with acrylamide isﬁlled with protein mixture containing 1῍ v/v of Parmalyte and 0.1῍ hydroxyetyl cellulose, then a voltageof ῌ25 kV is applied between the mobile-phase reservoir ﬁlled with catholyte (20-mM sodium hydroxide,pH 12.0) and the bu#er reservoir ﬁlled with an anolyte,
8῍ acetic acid solution (water῍methanol῍acetic acid,
50 : 42 : 8῍ (v/v/v); pH 3.0), for 15 or 20 minutes forfocusing. Next, gas pressure is applied to the cathodic
end, i.e., mobile-phase reservoir, while maintaining thevoltage, then the focusing proteins are pushed into thesample-introduction capillary and ionized.

1,035, which correspond to the 21-protonated moleculeof Mb and the 29-protonated molecule of CA II, respectively, in Fig. 2(a). After 9.2, 9.7, 11.7, and 12.3 minutesof pressuring the protein samples, clearly separated
peaks corresponding to the CA II (pI 5.4), CA II (pI 5.9),
Mb (pI 6.8), and Mb (pI 7.2) ions appeared, respectively.
The noises observed from 8 to 14 minutes were due toPharmalyte-containing compounds with a wide rangeof molecular weights. Mass spectra of CA II and Mb
obtained at 9.2 and 12.3 minutes are shown in Figs. 2(b)and (c), respectively. Multiply-charged ions of proteinsare clearly detected.
Figure 3 is an electrophorogram of the Mb and Cyt-c(pI 10.1) mixture. We injected 160 fmol of Mb andCyt-c (concentration of each solution was 0.1 mM), andῌ25 kV was applied to the mobile-phase reservoirs for
20 minutes, then a gas pressure of 0.20 kgf/cm2 wasapplied. We monitored the intensities at m/z 892 and814 corresponding to the 20-protonated molecule ofMb and the 15-protonated molecule of Cyt-c, respectively. After 5.6 and 9.3 minutes, peaks corresponding
to the Mb and Cyt-c ions appeared, respectively. Since
Pharmalyte molecules were concentrated around neutral in the pH gradient, the signal/noise ratios of theResults and Discussion
Figure 2 shows an electrophorogram of the protein
mixture (Fig. 2(a)) and mass spectra corresponding to
the peaks of the electrophorogram (Figs. 2(b) and (c)).
The sample mixture contained 0.6-mM CA II (pI 5.4), 0.6mM CA II (pI 5.9), and 1.2-mM Mb (mixture of pI 6.8 and7.2). And 940 fmol of each protein was injected into theseparation capillary. A voltage of ῌ25 kV was applied
to the mobile-phase reservoir for 15 minutes, then agas-pressure of 0.11 kgf/cm2 was applied to its surface.
We scanned the intensities at an m/z range from 600 to1,900 and monitored the intensity at m/z 847 andῌ 22 ῌFig. 2.

(a) Electrophorogram of carbonic anhydrase II(pI 5.4 and 5.9) and myoglobin (pI 6.8 and 7.2)
and mass spectra of (b) carbonic anhydrase II ata peak of 9.2 minutes and (c) myoglobin at apeak of 12.3 minutes in the electrophorogram.
The mobile-phase (inlet) reservoir and thebu#er (outlet) reservoir were ﬁlled with a 20mM sodium hydroxide (pH 12.0) in water and8῍ acetic acid solution (water῍methanol῍acetic
acid: 50 : 42 : 8῍, v/v/v; pH 3.0), respectively,
and the amount of injected samples was 940
fmol.
The separation capillary coated withacrylamide was 80 cm long and ῌ25 kV wasapplied between the two reservoirs for 15
minute, then a gas pressure of 0.11 kgf/cm2 wasapplied to the inlet reservoir.

CIEF Separation Combined with MS Using SSI for Protein Analysis

Mr. Yoshinobu Kohara, Mr. Tomoyuki Sakai, Mr.
Masao Kamahori, Dr. Tsuyoshi Sonehara, Dr. Kyoko
Kojima, Mr. Toshihiro Ishizuka, and Mr. Atsushi
Nemoto for their help in our experiments.
References
1)
2)
3)
4)
Fig. 3.

Electrophorogram of myoglobin and cytochrome-c (pI 10.1). The mobile-phase (inlet)
reservoir and the bu#er (outlet) reservoir wereﬁlled with 20-mM sodium hydroxide (pH 12.0)
in water and 8῎ acetic acid solution (water῍methanol῍acetic acid῍50 : 42 : 8῎, v/v/v, pH3.0), respectively, and the amount of injected
samples was 160 fmol. The separation capillary
coated with acrylamide was 80 cm long andῌ25 kV was applied between the two reservoirs for 20 minutes, then a gas pressure of 0.20
kgf/cm2 was applied to the inlet reservoir.

5)

6)

7)

8)

Cyt-c peak on the alkali side were higher than the Mbpeak.
In conclusion, CIEF-SSI/MS is valuable as a simple
and fast method for protein analysis. Furthermore, useof other types of mass spectrometers, for example,
time-of-ﬂight (TOF) and Fourier-transform ion cyclotron resonance (FT-ICR), as a detector of CIEF-SSI/MS,
will provide a powerful tool with high resolution andsensitivity.

9)
10)
11)
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13)

Acknowledgments
We thank Prof. Satoko Akashi of Yokohama CityUniversity, Prof. Koji Takio and Dr. Hisashi Kato ofRIKEN for their valuable discussions. We also thankDr. Keiko Kitagishi and Mr. Kazushi Sasa of Otsuka
Electronics Co., Ltd. for their valuable assistance andproviding the tools used in our study. We also thank14)

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Keywords: Capillary isoelectric focusing separation/mass
spectrometry (CIEF/MS), Sonic spray ionization, Protein,Proteomics

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