Efficient capillary electrophoresis of peptides and ...conformation. Such ‘nondenaturing’...

Chapter 6Capillary electrophoresis–mass spectrometry

of proteins at medium pH using

bilayer-coated capillaries

Catai, J. R.; Sastre Toraño, J.; de Jong, G. J.; Somsen, G. W.

to be submitted

Jonatan R. Catai

Efficient capillary electrophoresis of peptides and proteins with bilayer-coated capillaries 111

Summary

The feasibility of using noncovalently bilayer-coated capillaries for capillary

electrophoresis–mass spectrometry (CE–MS) of proteins was investigated using background

electrolytes (BGEs) of medium pH. The capillary was coated by successively rinsing the

capillary with solutions of the oppositely charged polymers polybrene (PB) and poly(vinyl

sulfonic acid) (PVS). Volatile BGEs containing ammonium formate and/or N-methyl

morpholine were tested at pH 7.5 and 8.5. Overall, these BGEs provided relatively fast

protein separations (analysis times of ca. 12 min) and showed high efficiencies (70,000–

300,000 plates) when the ionic strength was sufficiently high. Migration-time

reproducibilities were very favorable with RSDs of less than 1.0%. Infusion experiments

showed satisfactory MS responses for studied proteins dissolved in ammonium formate

(pH 8.5), however, high concentrations of N-methyl morpholine appeared to seriously

suppress the MS protein signals. Evaluation of the CE–MS system was performed by

analyzing a mixture of intact proteins yielding efficient separations and good-quality mass

spectra. CE–MS analysis of a reconstituted formulation of the biopharmaceutical

recombinant human growth hormone (rhGH) that was stored for a prolonged time, revealed

one degradation product, which was provisionally identified as desamido rhGH. Based on

the MS responses the amount of degradation was estimated to be ca. 25%.

Jonatan R. Catai

Efficient capillary electrophoresis of peptides and proteins with bilayer-coated capillaries112

6.1. Introduction

Capillary electrophoresis (CE) is a powerful separation technique, which shows

attractive characteristics for the analysis of biopolymers such as proteins [1-4]. In CE,

separations are based on differences in electrophoretic mobility, which is a function of

charge and size of the studied ionic species. When a protein undergoes degradation or

binds to other molecules (e.g. drugs), this might induce changes in the protein’s net charge

and/or shape, which may lead to altered electrophoretic migration. Separations in CE are

performed in open tubes without stationary phase and, therefore, analysis of proteins, in

principle, can be carried out under circumstances that do not significantly affect their

conformation. Such ‘nondenaturing’ conditions are difficult or impossible to achieve with

other separation methods like liquid chromatography (LC) and slab-gel electrophoresis.

Furthermore, CE can provide high separation efficiencies, relatively short analysis times

and requires only minute amounts of sample. Due to these characteristics, CE gradually

has become an essential separation technique for proteins [5], with applications in, for

instance, proteomics [6, 7], food analysis [8] and analysis of biopharmaceuticals [9-11].

Its significance is further illustrated by the recent introduction of CE methods in the

European Pharmacopoeia monographs of the pharmaceutical proteins erythropoietin (EPO)

[12] and human growth hormone (hGH, somatropin) [13].

CE analyses are normally performed using fused-silica capillaries. However,

particularly during protein analysis, interactions between the analytes and the inner capillary

surface can occur, leading to band broadening, unstable electroosmotic flow (EOF) and

poor migration-time reproducibilities. Therefore, in CE of proteins the use capillary coatings

is often advocated [1-5]. Recently, we have demonstrated the usefulness of capillaries

coated with a bilayer of the oppositely charged polymers polybrene (PB) and poly(vinyl

sulfonic acid) (PVS) for the analysis of peptides and proteins [14-16]. The PB-PVS coating

shows very stable migration times (RSDs < 1.0%) for these compounds when using BGEs

of various pH (2.5–9). This constancy in migration times is difficult to obtain with bare-

fused silica capillaries even with extensive capillary rinsing. Moreover, better separation

efficiencies (higher plate numbers) could be achieved with bilayer-coated capillaries.

Mass spectrometry (MS) has become an important technique for the analysis and

characterization of intact proteins and protein complexes [17-21]. Matrix-assisted laser

desorption ionization (MALDI) is frequently used for molecular weight determination of

proteins [22], but it is less suitable for coupling with flow systems. Electrospray ionization

(ESI) of proteins provides multiple charged ions with m/z values that often fall within the

range of conventional mass analyzers as quadrupoles and ion traps. Furthermore, ESI is a

Jonatan R. Catai


soft ionization method, which does not induce protein fragmentation and may even leave

the conformation of the analyzed protein unchanged [18, 23], which can be important

when structural integrity has to be probed. The combination of CE with ESI-MS clearly

provides a tool that can be very useful for purity and stability studies of proteins under

mild separation conditions. Until now, however, CE–MS of proteinaceous samples has

mainly been performed using acidic BGEs (pH < 4.5) in combination with bare fused-

silica capillaries or with capillaries coated with a positively-charged or neutral polymer [6,

24, 25]. So far, the use of medium-pH BGEs with coated capillaries for protein analysis by

CE–MS has been quite limited. Simó et al. [26] have used ammonium acetate BGE (pH

5.5) in combination with EPyM-DMA-coated (positively-charged polymer) capillaries for

the analysis of basic proteins in food products. He et al. [27] used Tris acetate (pH 6.0)

with capillaries coated with a neutral polymer for the analysis of degraded cytochrome c.

Quite a number of proteins have isoelectric points lower than 7 and, thus, carry a net

negative charge in medium pH buffers. Such proteins would preferably be analyzed by CE

using a negatively charged coating providing both electrostatic repulsion and a significant

electroosmotic flow (EOF). Hitherto, such CE–MS systems have not been described in

literature.

In this work, the potential of capillaries coated with PB-PVS for the CE–MS

analysis of proteins using volatile BGEs of medium pH was investigated. Recently, we

have demonstrated that PB-PVS coated capillaries can be used for CE–MS of peptides,

yielding highly efficient and reproducible separations [14]. In the present study, the influence

of the type, concentration and pH of the BGE on plate number, migration-time

reproducibility and MS signal intensity of proteins was examined. The performance of the

CE–MS system was evaluated using several test proteins and the biopharmaceutical

recombinant human growth hormone (rhGH). Finally, the usefulness of the system is

illustrated by the analysis of a solution of a commercial rhGH formulation, which had

been stored for a prolonged period of time.

6.2. Experimental

6.2.1. Chemicals

Polybrene (hexadimethrine bromide) and a 25% (m/v) aqueous solution of

poly(vinyl sulfonate) sodium salt (PVS) were from Sigma-Aldrich (Steinheim, Germany).

Jonatan R. Catai


A 1% (v/v) PVS solution was prepared by diluting the purchased PVS solution with Mili-

Q water (18.2 MÙ). Polybrene was dissolved to a final concentration of 1% (m/v) in Mili-

Q water. Formic acid was from Riedel-de Haën (Seelze, Germany). Ammonium hydroxide

(25%) was from Merck (Darmstadt, Germany) and N-methyl morpholine (99.5%) from

Sigma-Aldrich. The test proteins α-lactalbumin (bovine milk), insulin (porcine), and

carbonic anhydrase were from Sigma-Aldrich. Recombinant human growth hormone

(rhGH) protein (somatropin CRS) was from the European Directorate for the Quality of

Medicines (Strasbourg) and contained unspecified amounts of glycine, mannitol, lactose,

and sodium bicarbonate. Formulated rhGH (Humatrope, Eli Lilly, Houten, The Netherlands)

was reconstituted in water 18 months before the start of this study, and stored at 4 °C. The

resulting solution contained rhGH (1.5 mg/mL), glycine (1.5 mg/mL), mannitol (4.5 mg/

mL), disodium hydrogen phosphate (1 mg/mL), 0.3% metacresol, 0.29% glycerin and

glycerol. Stock solutions of test proteins (3 mg/mL) were weekly prepared in Mili-Q water

and stored at 4 °C. The insulin stock solution contained 0.2% acetic acid. A protein test

sample of insulin, carbonic anhydrase and α-lactalbumin was prepared by diluting aliquots

of stock solutions to the desired concentrations. rhGH CRS was diluted to 1.5 mg/mL with

Mili-Q water. BGEs were prepared by diluting ammonium hydroxide or N-methyl

morpholine to the desired concentration and the pH was adjusted to 7.5 or 8.5 with formic

acid. All BGEs were passed through a 0.22 µ m hydrophilic filter from Sartorius (Göttingen,

Germany) before use.

6.2.2. CE systems

Capillaries with an internal diameter (ID) of 50 µm were from Composite Metal

Services (The Chase, Hallow, UK). CE experiments with UV detection were performed

using a Beckman-Coulter (Fullerton, CA, USA) P/ACE MDQ capillary electrophoresis

instrument equipped with a diode array detector. Capillaries had a total and effective length

of 70 and 60 cm, respectively. Samples were injected for 7 s at 34.5 mbar (injection volume

of ca. 6 nL) and the separation voltage was 30 kV. The capillary was thermostated at 25 ºC

and absorbance detection of proteins was carried out at 214 nm.

CE–MS experiments were conducted using a PrinCE CE system from Prince

Technologies B.V. (Emmen, The Netherlands) applying capillaries with a length of 80 cm.

Samples were injected for 12 s at 35 mbar (ca. 9 nL). Injection conditions were such that

the sample zone was about 0.5% of the capillary volume to window. During sample injection,

the nebulizer gas flow and the electrospray voltage of the CE–MS interface were turned

Jonatan R. Catai


off. The separation voltage was 30 kV in all CE–MS experiments. Capillaries were

provisionally thermostated during CE–MS experiments by placing them inside a plastic

tube of ca. 2 mm ID with a length of ca. 60 cm that comprised most of the capillary, but not

the parts inside the auto-injector of the PrinCE and the sheath-liquid interface. In the middle

of the tube a tee piece was placed through which a constant flow of air (approx. 2 L/min)

of ambient temperature was led along the CE capillary in order to achieve effective heat

dissipation. In order to minimize hydrodynamic flow in the CE capillary induced by the

nebulizer gas, a reduced pressure in the range of -10 to -60 mbar was applied at the inlet

vial during CE–MS analysis [14]. MS infusion experiments were carried out by continuously

leading the solution of interest through the CE capillary at ca. 250 nL/min into the interface

and MS ion source with no separation voltage being applied.

6.2.3. Capillary coating procedure

New bare fused-silica capillaries were successively rinsed at 1400 mbar with 20

capillary volumes of water, 30 capillary volumes of 1 M NaOH, and 20 capillary volumes

of water. After this treatment, capillaries were coated by subsequently rinsing with 1.5

capillary volumes of a 1% PB solution at 35 mbar, 10 capillary volumes of water at 1400

mbar, 1.5 capillary volumes of a 1% PVS solution at 35 mbar, and 10 capillary volumes of

water at 1400 mbar. The capillary was then ready for CE analysis with the BGE of choice.

Between runs, coated capillaries were flushed at 1400 mbar with 4 capillary volumes of a

1% PVS solution. During rinses, PVS was prevented from entering the ion source by

opening the spray chamber and turning off the electrospray voltage.

6.2.4. MS system

CE was coupled to an Agilent Technologies 1100 Series LC/MSD XCT ion-trap

mass spectrometer (Waldbronn, Germany) equipped with an electrospray ion source via a

coaxial sheath-flow sprayer (Agilent Technologies). The CE capillary outlet was positioned

at 0.1–0.2 mm from the tip of the interface. Several types of sheath liquid (methanol–

water, isopropanol–water and acetonitrile–water) in several ratios (25:75, 50:50, 75:25)

and with various concentrations of formic acid (0, 0.5, 1, 5, 10%) were tested in order to

find stable spray conditions and optimum MS responses for the tested proteins. The optimal

sheath liquid appeared to be acetonitrile water formic acid (75:25:5, v/v/v) and was used

throughout this study. The sheath liquid was supplied by a syringe pump at 4 µL/min. The

nebulizer-gas pressure was 700 mbar and the flow and temperature of the drying gas were

Jonatan R. Catai


5 L/min and 200 °C, respectively. The electrospray voltage was 4.5 kV. MS detection was

carried out in the positive ion mode and each spectrum was an average of 2 scans. The ion

charge control (ICC) was set to 100,000 and the scanned mass range was 1000–2000 m/z.

Plate numbers of analyzed proteins were calculated using full width at half height as

measured from peaks observed in extracted-ion electropherograms. The instrument was

controlled by a PC running the LC-MSD data acquisition software from Agilent

Technologies. Charge assignment in the MS spectra was performed with the “Charge

deconvolution” utility available in the DataAnalysis V. 2.1 program of the Agilent software.

6.3. Results and discussion

6.3.1. Influence of BGE on separation and MS detection performance

In a previous study, we have demonstrated the suitability of PB-PVS coated

capillaries for reproducible and efficient analysis of proteins by CE–UV using BGEs of

Tris-phosphate (pH 7–8.5) [16]. In order to allow electrospray-mass spectrometric (ESI-

MS) detection of proteins, we investigated the performance of PB-PVS coated capillaries

in combination with volatile BGEs of medium pH. For this purpose, a test mixture of

insulin (50 µg/mL), α-lactalbumin, and carbonic anhydrase (200 µg/mL each) was analyzed

by CE–UV with various concentrations (25–150 mM) of ammonium formate (pH 7.5). An

improvement of separation efficiency (plate numbers) was observed with the increase of

the BGE concentration. However, repeated analyses with each BGE showed that the

migration times of the proteins consecutively decreased. This was most probably caused

by the lack of buffer capacity of the ammonium formate BGE at pH 7.5, as it was also

indicated by a significant pH difference between the BGEs present in the inlet and outlet

vial after only three runs.

The possibility of using a BGE of ammonium formate at pH 8.5 was then

investigated in the concentration range of 25–150 mM. CE–UV of the protein test sample

using the PB-PVS coated capillary showed an increase in plate numbers as the BGE

concentration was raised, reaching a maximum at 75 mM as it is depicted for α-lactalbumin

in Figure 6.1. Above this concentration, a decrease in plate numbers was observed for all

proteins. This decrease was possibly caused by an excess of Joule heating at high BGE

concentrations. This was further evidenced by repeated analysis (n = 5) of the test sample

at each BGE, which showed that migration-time RSDs increased from ca. 0.8% (BGE

concentration of 25–75 mM) to about 3% when higher BGE concentrations (100–150

mM) were used.

Jonatan R. Catai


Figure 6.1. Effect of the concentration of ammonium formate (pH 8.5) on the plate number of α-lactalbuminobtained by CE-UV using a PB-PVS coated capillary.

Next, the effect of the BGE concentration on the MS-signal intensity of the studied

proteins was examined. The test proteins α-lactalbumin, insulin and carbonic anhydrase

were each dissolved in water and in various concentrations of ammonium formate (pH

8.5). These solutions were infused into the sheath-liquid interface through a PB-PVS coated

capillary. Upon increasing the BGE concentration, a decrease in MS signal intensity of all

proteins was observed. This trend is illustrated for α-lactalbumin in Figure 6.2. For CE–

MS analysis at pH 8.5, a BGE of 75 mM ammonium formate was selected as a compromise

between plate number, migration-time reproducibility and MS response for the studied

proteins.

Figure 6.2. Effect of the concentration of ammonium formate (pH 8.5) on the MS signal intensity of α-lactalbumin obtained during infusion experiments.

Jonatan R. Catai


In order to be able to perform CE–MS of proteins with a buffering BGE at physiological

pH, the possibility of using of N-methyl morpholine (pKa 7.4) instead of ammonium

formate was examined. The protein test mixture was analyzed by CE–UV using bilayer-

coated capillaries with various concentrations (25–200 mM) of N-methyl morpholine

adjusted to pH 7.5 with formic acid. Plate numbers for the proteins were quite

unsatisfactory at 25 mM N-methyl morpholine, but increased for higher concentrations

reaching a plateau of about 150,000 at 100 mM. Migration-time reproducibilities were

quite favorable (RSDs < 1.0%), especially in the 25–100 mM range. However, MS

infusion experiments revealed that the N-methyl morpholine caused serious suppression

of the protein ionization. A decrease in MS signal intensity of 25–80% was observed as

N-methyl morpholine concentrations were raised from 25 to 200 mM. In order to come

to a BGE at pH 7.5 with sufficient ionic strength and buffer capacity, but still acceptable

MS sensitivity, the feasibility of a BGE comprising a low concentration of N-methyl

morpholine (20 mM) and a high concentration of ammonium formate (75 mM) was

investigated. CE–UV of the protein test sample revealed plate numbers of 100,000–

300,000 for the proteins and excellent migration-time reproducibilities (RSDs < 1.0%).

After five runs, no significant alteration of the pH values of the BGE solutions in the

inlet and outlet vial was observed. These results show that the tested BGE had the required

ionic strength and a good buffer capacity. MS infusion experiments with this BGE showed

that the MS signals for the studied proteins were ca. 20% lower than the signal intensities

obtained with a BGE of 75 mM ammonium formate (pH 8.5).

Finally, CE–MS of proteins using PB-PVS coated capillaries was evaluated

with an ammonium formate BGE (pH 8.5) of 75 mM as it provided optimum signal

intensity for proteins. A test sample of insulin (50 µg/mL), carbonic anhydrase (400 µg/

mL) and α-lactalbumin (1 mg/mL) was analyzed with the CE-MS system (Figure 6.3).

Plate numbers ranged from 70,000 to 100,000, which is quite favorable for CE–MS of

proteins. Still, especially the plate numbers obtained for carbonic anhydrase and α-

lactalbumin were lower than those obtained with CE-UV. These lower plate numbers

were found to be caused by the relatively high protein concentrations in the sample,

which induced some extra band broadening. The higher concentrations were necessary

due to the limited detection sensitivity of the ion-trap mass spectrometer obtained for

these proteins. Actually, CE–UV analysis of the same sample revealed similar plate

numbers for the proteins. Migration times of the proteins with CE–MS were very

reproducible with RSDs of less than 1.0% obtained for five consecutive runs. From the

acquired mass spectra (Figure 6.3B-D) the molecular masses of the analyzed proteins

Jonatan R. Catai


were determined to be 29022, 5807 and 14187 Da for carbonic anhydrase, insulin and α-

lactalbumin, respectively, which nicely matches the expected values. These results

demonstrate that PB-PVS coated capillaries can be used for the CE–MS of intact proteins,

yielding efficient and reproducible separations. Furthermore, it can be concluded that

the sheath-liquid interface does not cause significant broadening of the protein peaks

produced by the CE system. This is in line with our previous work in which plate numbers

for peptides analyzed by CE–MS were similar to those obtained with CE–UV [14].

6.3.2. CE–MS of human growth hormone

The potential of the bilayer capillary coating for CE–MS of proteins at medium

pH, was further tested by the analysis of the protein recombinant human growth hormone

(rhGH). rhGH is a biopharmaceutical that is used in the treatment of retarded growth

and dwarfism caused by the inadequate production of the hormone during the growth

period. Recently, the European Pharmacopoeia (Ph. Eur.) introduced a method for the

analysis of this protein by CE with UV detection using bare fused-silica capillaries [12].

This method, however, is not MS compatible as it uses high concentrations of non-

volatile phosphate buffer as BGE. Meanwhile, we have also demonstrated that faster

and more reproducible CE analysis of rhGH can be achieved with bilayer-coated

capillaries. These results will be published elsewhere [28].

First, a sample containing 1.5 mg/mL of rhGH (somatropin CRS) was analyzed by

CE–MS using a PB-PVS coated capillary with a BGE of 75 mM ammonium formate (pH 8.5).

The sample yielded one symmetric peak at ca. 10.8 min with a plate number of about 90,000.

Deconvolution of the acquired mass spectrum resulted in an estimated molecular mass for

rhGH of 22124 Da, which is in line with reference data. Subsequently, in order to test the

feasibility of the CE–MS system for protein degradation studies, a reconstituted commercial

rhGH formulation was analyzed. The rhGH solution had been stored for 18 months at 4 °C.

Notably, this time is far beyond the storage period (14 days at 2-8 °C) for reconstituted rhGH

recommended by the supplier. Figure 6.4 depicts the CE–MS result, which clearly shows a

degradation product. Deconvolution of the mass spectra of the main and minor peaks shows

that both constituents have virtually identical masses, i.e., 22124 Da. This is an indication that

the protein may have undergone deamidation. This degradation route involves the transition of

an asparagine residue into an aspartic acid residue leading the protein to a gain of one negative

charge at neutral pH. CE is particularly useful to reveal charge modifications, as the

electrophoretic mobility depends on the charge-to-size ratio of a protein. The change of only

one charge already leads to a clear shift in migration time. However, upon deamidation the

Jonatan R. Catai


Figure 6.3. CE–MS of a protein test mixture using a PB-PVS coated capillary and a BGE of 75 mMammonium formate (pH 8.5). (A) sum of extracted-ion electropherograms obtained at m/z 1383.3,1452.7 and 1419.5; (B), (C) and (D) average mass spectra of peaks 1 (carbonic anhydrase), 2(insulin) and 3 (α-lactalbumin), respectively.

Jonatan R. Catai


overall mass change of the protein is only 1 Da, which explains the detected masses of both

compounds to be practically the same. The limited mass resolution of the ion-trap mass

spectrometer does not permit the detection of such a small mass difference. For that, a mass

analyzer with higher mass resolution such as time-of-flight (TOF) should be used. Based on the

MS responses the percentage of degradation of the rhGH product was estimated to be ca. 25%.

Figure 6.4. CE–MS of a formulation of rhGH which after reconstitution was stored for 18 months at 4 °C. APB-PVS coated capillary was used with a BGE of 75 mM ammonium formate (pH 8.5). (A) sumof extracted-ion electropherograms obtained at m/z 1476.0 and 1581.2; (B) and (C) averagemass spectra of peaks 1 (intact rhGH) and 2 (degradation product), respectively.

The possibility of using 20 mM N-methyl morpholine with 75 mM ammonium

formate (pH 7.5) as BGE for the CE–MS analysis of the degraded rhGH formulation was

also examined. The resulting separation profile (Figure 6.5) and plate numbers were

comparable to those obtained with the BGE with pH 8.5. The analysis time showed to be

slightly longer at pH 7.5, which was probably caused by the somewhat higher ionic strength

of the morpholine-ammonium formate BGE. As expected, CE–MS using the morpholine-

ammonium formate BGE presented a lower signal-to-noise ratio for rhGH due to increased

ionization suppression and a higher background noise caused by N-methyl morpholine.

However, significant signals were still obtained allowing the effective profiling of the

sample at pH 7.5. Deconvolution of the mass spectra of each acquired peak, once more

revealed the same molecular mass (22124 Da), suggesting that peaks corresponded to

intact and desamido rhGH.

Jonatan R. Catai


Figure 6.5. CE–MS of a formulation of rhGH which after reconstitution was stored for 18 monthsat 4 °C. A PB-PVS coated capillary was used with a BGE of 20 mM N-methylmorpholine with 75 mM ammonium formate (pH 7.5). The depicted trace is the sumof extracted-ion electropherograms obtained at m/z 1476.0 and 1581.2. Peaks: 1, intactrhGH; 2, degradation product.

6.4. Conclusion

The usefulness of PB-PVS coated capillaries for CE–MS analysis of proteins

using volatile BGEs of medium pH is demonstrated. Favorable plate numbers and very

good migration-time reproducibilities were achieved for the studied proteins in relatively

short analysis times. The use of BGEs with sufficient buffer capacity and relatively high

ionic strength were important in order to obtain good separation performance. A BGE of

ammonium formate alone appeared to be suitable for analysis at pH 8.5. However, the

addition of N-methyl morpholine to the BGE was required in order to achieve both

appropriate buffering and MS compatibility at pH 7.5. The potential of the PB-PVS system

for protein CE–MS was indicated by the analysis a degraded formulation of rhGH using

the BGEs at pH 7.5 and 8.5. The obtained CE and MS data suggests that the rhGH has

undergone deamidation. The favorable migration-time reproducibilities induced by the

bilayer coating can be of great importance for the comparison of CE–MS profiles obtained

in time, e.g., during stability studies of pharmaceutical protein. Further characterization of

Jonatan R. Catai


proteins by CE–MS would profit from the use of mass spectrometers, such as TOF as they

can yield higher mass resolution than the used ion-trap mass spectrometer.

Acknowledgement

The authors thank Peter M.J.M. Jongen from the Center for Biological Medicines

and Medical Technology of the National Institute for Public Health and the Environment

(Bilthoven, The Netherlands) for useful advice and stimulating discussions.

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