CESI-MS of Intact Proteins
Rob Haselberg,1 Chitra K. Ratnayake,2 Gerhardus J. de Jong,1 and Govert W. Somsen1
1 Biomolecular Analysis, Utrecht University, The Netherlands
2 SCIEX, Brea, CA, USA

Introduction
There is a growing need for selective and sensitive analyticalA

stainless-steel capillary
polyimide

tools for the characterization of intact proteins. The effective
coupling of capillary electrophoresis and electrospray ionization{

time-of-flight mass spectrometry (CE-ESI-TOF-MS) may offer an
attractive option by providing both the separation and detection
required to distinguish structurally related protein species.1,2 In
order to bring CE-ESI-MS to a high performance level, novel

fused-silica wallCEcapillary

sheathless CE-MS interfacing was studied to achieve sensitive
detection of intact proteins.
Based on a design of Moini,3 a prototype CESI sprayer wasrecently developed in the laboratories of Beckman Coulter,
Inc. Flow rates in CE are very low at nanoliter per minute, andthe initial droplets formed during the sheathless electrosprayprocess are small, which leads to more efficient ionization (i.e.,nanospray). With sheathless1 interfacing, the ESI [2] sprayelectrical contact

conductiveliquidB
static conductive
liquid feed

tip can be positioned close to the MS inlet, thereby improving

porous tip(length, ~4 cm;wall thickness, 5 µm)

nanospray
endplate

MS inlet

spray tip

ion sampling efficiencies. Overall, this can lead to enhanced

XYZstagesensitivity and lower limits-of-detection (LODs). The CESI
sprayer provides electrical contact without the need for metal
conductive coating, microelectrodes or liquid junctions. In theCESI design (Figure 1), the last 3–4 cm of the bare fused-silicacapillary are etched with hydrofluoric acid until the section
becomes conductive, producing a ~5 μm thick porous wall, whichis conductive when in contact with an electrolyte. Capillaries withporous tips are simply inserted in a stainless-steel ESI needle
filled with a static conductive liquid.
In this study, the performance of the CESI-MS of intact proteins
was evaluated using some model proteins. Reproducibility,linearity and LODs were established. It is demonstrated that
CESI-MS provides enhanced responses for intact proteins
resulting in sub-nM detection limits. This is a 50- to 140-foldimprovement compared to conventional sheath-liquid CE-MS
interfacing using the same capillary dimensions.

CE capillary

Figure 1. The PA 800 Plus System.

Materials and methods
Chemicals: Acetic acid (99.8%), ammonium hydroxide (25%)
and isopropanol (IPA) were obtained from Merck. Insulin(bovine pancreas), carbonic anhydrase II (bovine erythrocytes),ribonuclease A (bovine pancreas) and lysozyme (chicken eggwhite) were from Sigma-Aldrich. Protein test mixtures wereprepared in the appropriate concentration with deionized water.
A background electrolyte (BGE) of 100 mM acetic acid (pH 3.1)
containing 5% IPA was prepared by diluting 0.171 mL glacialacetic acid to 30 mL with deionized water/IPA (95/5, v/v) andadjusting the pH with ammonium hydroxide.

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CE system: Experiments were carried out on a Beckman Coulter

determination of detection linearity and LOD, extracted-capillary electrophoresis instrument. The separation voltage

ion electropherograms (EIE) for the 4 model proteins werewas -30 kV and the capillary temperature was 20 °C. Fused-constructed from their most abundant m/z signals. These weresilica capillaries (total length, 100 cm; inner diameter, 30 μm;

m/z 1147.7 and 1434.1 for insulin; m/z 1210.4, 1262.9, 1320.3,

outer diameter, 150 μm) equipped with the porous tip [3] (length,1383.1 and 1452.2 for carbonic anhydrase II; m/z 1521.2, 1711.1

3–4 cm) were supplied by Beckman Coulter. The capillaries

and 1955.5 for ribonuclease A and m/z 1431.6, 1590.34 andwere precoated with polyethylenimine (PEI). At the beginning of1789.0 for lysozyme.

each day, the coated capillary was conditioned by flushing thecapillary at 50 psi with air (10 min), methanol (20 min), deionizedwater (5 min) and BGE (10 min). Before each run, the capillary
was flushed for 3 min (50 psi) with fresh BGE. The sample wasResults and discussion
The porous tip capillary was placed in a grounded needle that

injected for 10 s at 5 psi.

was positioned on an XYZ-stage (Figure 1B). The electrical

CE-MS: MS detection was performed using a Bruker Daltonics

BGE. The liquid hardly evaporated from the needle and the samemicrOTOF orthogonal-accelerated time-of-flight (TOF)

contact was established by filling the stainless-steel needle withmass spectrometer.
Sheathless CE-MS: The capillary with the porous tip was placedin a grounded stainless-steel needle that could be positioned by
an XYZ-stage (Beckman Coulter) fitting the Bruker micrOTOF
instrument (Figure 1B). A nanospray endplate and gas diverter
were installed to allow nano-electrospray ionization (nano-ESI).
The porous tip protruded the grounded needle approximately 0.5
cm and the needle was filled with BGE to establish the electricalcontact. The optimized spray conditions were as follows: dry gas
temperature, 180 °C; dry gas nitrogen flow, 3.0 L/min; nebulizer
pressure, 0.0 bar. Electrospray in positive ionization mode wasachieved using an ESI voltage of -2.1 kV.

Important:• A separation current above 5 μA might cause permanent
damage to the separation capillary.
• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

Figure 2. (A) BPE obtained with sheathless CESI-MS of a mixture of (1)insulin; (2) carbonic anhydrase II; (3) ribonuclease A and (4) lysozyme(each 5 μg/mL). (B) Mass spectra obtained at the apices of peaks 1–4.
For further conditions, see the materials and methods section.

liquid could be used for an entire day of measurements. The
porous tip capillaries were coated with positively charged PEI,
which exhibits a net positive charge when an acidic BGE is used.
Under these conditions, adsorption of peptides and proteins tothe capillary wall will generally be avoided due to electrostatic

Sheath-liquid CE-MS: For sheath-liquid interfacing, the capillary

repulsion. Most stable electrospray formation and highest analytewith porous tip was placed in a grounded coaxial CE-MS sprayer

signals were obtained with the capillary tip at a distance of(Agilent Technologies) installed on a conventional ESI source

3 mm to the MS inlet.

comprising the standard ESI endplate and capillary cap. A flow of
2 μL/min of IPA-water-acetic acid (75:25:0.1, v/v/v) was applied
as sheath liquid. The optimized conditions for sheath-liquidinterfacing were: dry gas temperature, 180 °C; dry gas nitrogenflow, 4 L/min; nebulizer pressure, 0.4 bar; ESI voltage, -4.0 kV.

A mixture of insulin, carbonic anhydrase II, ribonuclease A andlysozyme was analyzed by sheathless CESI-MS. Using a BGE
of 100 mM acetic acid (pH 3.1) with 5% IPA, the 4 proteins werebaseline separated within 11 min (Figure 2A). IPA was added tothe BGE as it provided a modest gain in protein signal intensities.

Data analysis: CESI-MS data were analyzed using Bruker

Good quality mass spectra were obtained under these conditions

Daltonics data analysis software. Base-peak electropherograms(Figure 2B). Deconvolution of the mass spectra yielded masses(BPE) were constructed in the range m/z 1000–3000. Forof 5733.6 Da (insulin), 29024.6 Da (carbonic anhydrase II),

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13681.8 Da (ribonuclease A) and 14304.5 Da (lysozyme),

Protein signals obtained with sheath liquid interfacing wererespectively, which agreed well with their expected molecular

lower by a factor of approximately 10 to 50 with respect tomasses. The overall performance of the sheathless CESI-MS

CESI-MS (cf. Figure 2A and Figure 3; note that injected protein

system was further evaluated by assessing the repeatability,concentrations are 5 and 50 μg/mL, respectively). In particular,detection linearity and LODs for the test proteins. Migration time

insulin showed reduced response with sheath-liquid CE-MS.

RSDs for all 4 proteins were less than 0.8% (n=5) and peak

The lower protein signals are the overall result of the dilution ofarea RSDs were within 8% (n=5). Plate numbers ranged fromthe CE effluent by the sheath liquid and the lower ionization and

0.5 x 105 (insulin) up to 1.5 x 105 (ribonuclease A). Clearly, theion sampling efficiencies obtained using conventional ESI instead

CESI-MS system allows repeatable and efficient analyses ofof nanospray. It should be noted that the IPA in the sheath liquid

intact proteins. To check for detection linearity, protein mixturesstrongly enhances protein ionization, as without IPA, proteinwith concentrations between 0.05 and 25 μg/mL of each protein

signals are even much lower. Furthermore, the baseline noise

were prepared and each solution was analyzed in triplicate. For

observed is more favorable in CESI-MS (Figure 4).

all proteins, good linear relationships (Table 1) between injected

The application of sheath liquid causes significant chemical

concentration and obtained peak areas were obtained. Table 1

noise, which is avoided with CESI-MS. Overall, the LODs

also lists the LODs (S/N=3) achieved with sheathless CESI-MS.

achieved with CESI-MS are considerably lower than for

Sub-nM levels could still be detected for carbonic anhydrase II,

sheath-liquid CE-MS (Table 1).

ribonuclease A and lysozyme, indicating very favorable LODs for
CESI-MS of intact proteins.

Conclusion

In order to assess the gain in signal provided by CESI-MS, a

The performance of CESI-MS for the analysis of intact proteinscomparison with sheath-liquid CE-MS was made. The same

was evaluated. Sub-nanomolar LODs were obtained with thecapillary (i.e., equipped with the porous tip) was placed in a

CESI-MS system using a nanoESI source. These are highly

coaxial sprayer installed on a conventional ESI source. A sheathfavorable sensitivities that, to our knowledge, have not beenliquid of IPA-water-acetic acid (75/25/0.1, v/v/v) was applied at aachieved before with CE-MS for intact proteins. The gain inflow rate of 2 μL/min. These conditions had shown to be optimal

performance of the CESI-MS system with respect to sheath-for intact protein analysis in a previous CE-MS study.4 A baselineliquid CE-MS can be attributed to the reduced noise levels andseparation within 10 min (Figure 3) and linear protein signals

increased analyte responses. The very favorable LODs and(Table 1) were obtained for the test proteins, indicating the proper

overall performance indicates that CESI-MS can be highly usefulfunctioning of the sheath-liquid interfacing.

Proteinfor intact protein analysis.
Sheathless interfacec

Sheath liquid interfaced

R2LOD

R2LOD

insulin

0.999

1.28

0.992

106

carbonic anhydrase II

0.989

0.58

0.981

79

ribonuclease A

0.992

0.62

0.989

33

lysozyme

0.997

0.50

0.990

41

Table 1. Linearity (R2)a and LODs (nM)b for the 4 model proteins obtained with CESI-MS and CE-MS with sheath-liquid interfacing.
a. Concentration range, 0.05–25 μg/mL (CESI-MS) and 1–100 μg/mL (sheath liquid).
b. Concentration to yield S/N ratio of 3 as calculated by extrapolation from 1-μg/mL injection. For carbonic anhydrase analyzed with sheath-liquid
CE-MS, a 5-μg/mL injection was used.
c. BGE, 100 mM ammonium acetate (pH 3.1) containing 5% (v/v) isopropanol.
d. BGE, 100 mM ammonium acetate (pH 3.1).

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Figure 4. Baseline signal and noise in the 0.9–5.9 min interval during(A) CESI-MS and (B) sheath-liquid CE-MS of the protein test mixture.
Traces represent extracted-ion traces for (1) insulin (m/z 1434.1); (2)carbonic anhydrase II (m/z 1262.9); (3) ribonuclease A (m/z 1711.1)
and (4) lysozyme (m/z/ 1590.3). For further conditions, see the materials
and methods section.

Figure 3. BPE obtained with sheath-liquid CE-MS of a mixture of (1)insulin; (2) carbonic anhydrase II; (3) ribonuclease A and (4) lysozyme(each 50 μg/mL) using a conventional ESI source and a sheath liquid of
isopropanol-water-acetic acid (75/25/0.1, v/v/v). For further conditions,see the materials and methods section.

References
1. Haselberg, R.; de Jong, G.J.; Somsen, G.W. J. Chomatogr.
A. 2007, 1159, 81.
2. Haselberg, R.; de Jong, G.J.; Somsen, G.W. Electrophoresis.
2011, 32, 66.
3. Moini, M. Anal. Chem. 2007, 79, 4241.
4. Haselberg, R.; de Jong, G.J.; Somsen, G.W. Anal. Chim Acta.
2010, 678, 128.

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