High Resolution Intact Glycoprotein Analysis by CESI-MS
Scott Mack, Hans Dewald, Gina Donvito, Kimberly Kwong, Chitra Ratnayake
Beckman Coulter, Inc.; Discovery Solutions Business Center, Brea, CA, USA
Figure 5a.

OptiMS CESI Interface

Introduction

20101014_TEA9_Run5 835 (14.215)

TOF MS ES+
3.99e414898.00

100

The analysis of intact proteins by Electrospray Ionization - Mass
Spectrometry (ESI-MS) provides the most direct route for theidentification and characterization of these highly complex
molecules. With the potential presence of multiple isoforms, it is also
widely accepted that the presence of a liquid-phase separation
technique upstream from the MS detection step would greatly
contribute to a complete deciphering of the structure of thesemolecular entities. Capillary electrophoresis (CE), compatible withthe preservation of delicate protein structure, post-translational andother modifications, while simplifying sample complexity throughhigh resolution separation capabilities, appears as a very valuable
alternative prior to introduction into the mass spectrometer. To
achieve the greatest advantage of coupling CE with ESI-MS for
intact protein analysis, we are currently developing a novel
technology called CESI that integrates CE with ESI into a single
dynamic process. The CESI technique has been successfully
implemented for the analysis of a model glycoprotein, Ribonuclease
B (RNase B). The efficient coupling between these liquid and gas
phase separation systems allowed the rapid, sensitive and highresolution detection of the five known Ribonuclease B glycoforms.
Further deconvolution of the resulting Time of Flight (ToF) mass
spectra by the Maximum Entropy Algorithm identified differences inthe attached high mannose structure as the source of theelectrophoretic heterogeneity. Produced on multiple sheathlesssprayers, the data confirm that these separations are highly
repeatable and reproducible.

14898∆162 Da

15060%

15060.00

15384
15384.00

15222
15546

15222.00

15546.00

Figure 1. The terminal portion of the OptiMS capillary has been made014700

porous to ion flow, providing an electrical contact between the BGE
inside the separation capillary and a conductive liquid filling a metal ESI
needle. This porous glass junction provides the ground for the CE circuitand high voltage contact for the ESI circuit .

14800

14900

15000

15100

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15300

15400

15500

mass
15700

15600

Figure 5b.
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14898.00

100

20110324_DTEA78_PTMPLus_Run3 708 (12.056)

13682

13682.00

100

14898

TOF MS ES+
TOF 2.68e4
MS ES+
2.23e5%∆1216 Da%

15060.00

RNase A
015384.00

13300

13400

13500

13600

13700

13800

13900

14000

14100

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mass
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Materials and Methods
Instrumentation

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15546.00

All CE-MS separations were performed on a prototype CESI 8000 High
Performance Separation - ESI Module*. Separations were performed onOptiMS 100 cm long 150 µm o.d. x 30 µm i.d. Surface+ coated capillaries.
Instrument temperature settings were 10 ºC for the sample storage moduleand the OptiMS capillary was maintained at 20ºC with a rapidly circulating
liquid coolant.
MS detection was performed using a Waters XevoŦ QToF set with ascanning range between 800 – 3000 m/z and a sampling rate of 1 Hz.
Electrospray ionization was achieved at 1.4 – 1.6 kV with the CESI 8000
OptiMS sprayer needle filled with background electrolyte (BGE).

14800

that the inner lumen of the capillary is preserved and the rim is highly
sharpened to reduce the outer diameter radius. The shape is ideal for
the ESI at low flow and electrospray voltages.

Reagent
MeOH
MeOH
AirAir1

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1)
2)
3)
4)
5)
6)

Haselberg, R., Ratnayake, C. K., de Jong, G. J., Somsen, G. W., J. Chromatogr. A., 2010, 1217, 7605 – 7611.
Garza, S., Moini, M., Anal. Chem. 2006, 78, 7309 – 7316.
Cloupeau, M., Prunet-Fosh, B. J. Electrost. 1989, 22, 135-159.
Karina, M., Bones, J., Kattla, J.,J., Rudd, P.,M., N.Chem.Bio. 2010, Vol 6, 713 – 723.
Marginean, I., et al., Anal. Chem. 2007, 79, 8030-8036.
Pramanik, B. N., Ganguly, A. K., Gross, M., Applied Electrospray Mass Spectrometry, Practical Spectroscopy SeriesVol. 32, Dekker, M. CRC Press Inc., Boca Raton, FL 2002, pp. 101–137.

16000

16200

16400

16600

mass

16800

TOF MS ES+1111.6+1196.9+1296.6+1414.4+1555.7+1728.5+1944.5
9.09e3

Man 5 Hex5HexNAC2

Man 6 Hex6HexNAC2TOF MS ES+BPI
778

100

Man 7 Hex7HexNAC2%81. Bovine α Lactalbumin Type III 14177 Da
2. Human Carbonic Anhydrase I 28781 Da

63

Man 8 Hex8HexNAC27

3. Bovine α -Chymotrypsinogen A Type II 25656 Da
4. Bovine β-Lactoglobulin A 18363 Da
5. Bovine β-Lactoglobulin B 18276 Da

Man 9 Hex9HexNAC2

6. Bovine Ribonulcease A Type 1-A 13682 Da
013.45%

7. Chicken Lysozyme 14304 Da
8. Bovine Cytochrome C 12229 DaTime
13.50

13.55

13.60

13.65

13.70

13.75

13.80

13.85

13.90

13.95

14.00

14.05

14.10

14.15

14.20

14.25

14.30

Figure 6. Shows the assignments of the various high mannose structures
to the five peaks present in the separation of RNase B. The shift inelectrophoretic mobility is also consistent with the addition of mass.

24 5

Peak Area Percent
Avg.
Man 5
41.76%
Man 6
27.27%
Man 7
7.97%
Man 8
16.58%
Man 9
6.48%

7*0Time
7.50

8.00

8.50

9.00

9.50

10.00

10.50

11.00

11.50

12.00

12.50

13.00

Figure 3. Eight proteins (50 µg/mL each) of various intact masses andisoelectric points are well separated using a 100 mM ammonium acetate pH
3.1 buffer at 20 kV reverse polarity. Partial separation between the two formsof Bovine β-Lactoglobulin show the high resolution of the system. The twopeaks attributed to Lysozyme are not differentiated by the Mass
Spectrometer indicated by the same intact mass, but can be easily separated
by the CE, indicating a difference between the two components.

TOF MS ES+1111.6+1196.9+1296.6+1414.4+1555.7+1728.5+1944.5
9.09e3100

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Peak 4•

Surface+ Coated OptiMS capillaries are capable of separating
and detecting a broad range of proteins.

•Separations of RNAse B produced five highly resolved peaks
which were identified as high mannose glycoforms.

•

Highly reproducible assessment of the percent composition
were achieved with RSD ranging between 3.65% to 8.45%

Peak 3
Peak 2

Peak 1

013.45Conclusions

TOF MS ES+
1111.6+1196.9+1296.6+1414.4+1555.7+1728.5+1944.5
9.09e3

Peak 5

100RSD
3.65%
3.92%
8.45%
5.74%
6.43%

Table 1. Shows the results of 100 compositional analysis runs ofGlycoprotein Analysis by CESI 8000
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StdDev
1.52%
1.07%
0.67%
0.95%
0.42%

RNase B glycoforms performed on 3 separate capillaries
over 5 days. The results show that the analysis method are both
highly repeatable and reproducible.

Protein Analysis Procedures

References

15800

Protein Analysis by CESI 8000

Capillary Maintenance Procedures

Prior to starting an analysis cycle, the OptiMS sprayer needle was filledwith conductive by applying 50 psi on a vial in the buffer outlet tray
containing BGE for 1 min. The cationic coating was conditioned byapplying 50 psi on a vial in the buffer inlet for 10 min., then at 20 kV inreverse polarity for another 10 min.
Protein samples were hydrodynamically injected into the separation
capillary using 5 psi for 5 sec. for a total plug volume of approximately 7 nL
and separated using a 20 kV potential in reverse polarity. A separate BGE
vial was used to rinse the capillary for 6 min. at 50 psi following thecompletion of the separation.

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100

Pressure Direction Duration
50 psiForward 60 min.
50 psi
Reverse 60 min.
50 psiForward 10 min.
50 psi
Reverse 10 min.

Newly coated capillaries were cleaned, and used capillaries were preparedfor storage following the completion of an analytical cycle by first purging
the separation capillary and conductive solution capillary for 5 min. at 50
psi with MeOH, then electrokinetically cleaned at 20 kV in reverse polarity.
After cleaning the capillaries, both capillaries were air-dried using pressure
rinses with empty vials for 5 min. at 50 psi.

15400

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The reagents used are as follows: OptimaŦ LC/MS grade methanol(MeOH) (Fisher Scientific P/N 456-1), 0.1 M sodium hydroxide (NaOH)(Beckman Coulter P/N 391988), 0.1M hydrochloric acid (HCl) (BeckmanCoulter P/N 391646).
Prior to introducing the Surface+ Capillary Coating Reagent (Beckman
Coulter P/N B08746), the capillary was conditioned by performing a seriesof conditioning step washes.

Following the conditioning step, a 20% mixture of Surface+ Coating
Reagent in MeOH was introduced into the capillary at 50 psi for 85 min.
The coating reagent was left to incubate in the capillary overnight to allow
for the covalent attachment between the capillary wall and the cationicpolymer in the Surface+ coating reagent. After overnight incubation, thesystem was purged of the Surface+ coating reagent using a series ofpurge steps.

15200

electropherogram in Figure 4 show a mass shift of 162 Da, this along withthe 1216 Da shift between the glycosylated (RNaseB) and non-glycosylated(RNase A) provides strong evidence that the peaks observed in theelectropherogram are the result of an attached high mannose structure
containing between 5 and 9 mannose subunits.

Figure 2. SEM imaging of the OptiMS Capillary emitter tip shows

A 200 mM acetic acid solution was produced by diluting 5.7 mL of 17.4 M
glacial acetic acid (EMD P/N UN 2789) in 494.3 mL of 16 MΩ water using
a 500 mL volumetric flask. The 200 mM acetic acid solution was titrated toa pH of 3.6 with a 200 mM ammonium acetate solution produced bydiluting 2.7 mL of 7.5 M ammonium acetate (Sigma P/N A2706-1L) in 97.3
mL of 16 MΩ water using a 100 mL volumetric flask.

Reagent
Pressure Direction Duration
MeOH
50 psiForward 20 min.
16 MΩ Water 50 psi
Forward 10 min.
0.1 M NaOH
50 psiForward 20 min.
0.1 M HCl50 psi
Forward 10 min.
16 MΩ Water 50 psi
Forward 10 min.
MeOH50 psi
Forward 20 min.
Air50 psi
Forward 20 min.

15000

Figures 5 a, b: The calculated intact mass between the peaks in the

200 mM Ammonium Acetate pH 3.6 BGE Solution

Capillary Coating Procedure

RNase B

0Time
13.50

13.55

13.60

13.65

13.70

13.75

13.80

13.85

13.90

13.95

14.00

14.05

14.10

14.15

14.20

14.25

14.30

0Time
1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

16.00

Figure 5. Separation of (200 µg/mL) Bovine Ribonuclease B at 20 kV* In development.
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For Laboratory Use Only; not for use in diagnostic procedures.
Beckman Coulter and the stylized logo are trademarks of Beckman Coulter, Inc. and are registered with theUSPTO. CESI and OptiMS are trademarks of Beckman Coulter, Inc.

reverse polarity in 200 mM ammonium acetate pH 3.6 buffer resulted in theseparation of five distinct components.
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