A covalent, cationic polymer coating method for theCESI-MS analysis of intact proteins and polypeptides
Key Benefits:Easy 3-step covalent coating procedureEfficient, robust, reproducible separation of intact proteins, using positively-charged coating

High efficiency electrospray ionization in the low-nanoflow regime



CESI capillary can be re-coated

Marcia R Santos, Chitra K. Ratnayake, Bryan Fonslow, Andras Guttman
SCIEX Separations, Brea, CAIntroduction
Both capillary electrophoresis (CE) and mass spectrometry (MS)Intact protein separations when using LC columns or bare-fused

are powerful techniques for intact protein analysis, particularly forsilica capillaries are known to suffer from the adsorption andthe characterization of biologics and, more specifically,poor surface desorption characteristics of some proteins. Whiletherapeutic monoclonal antibodies (mAbs). While comprehensive

poor protein desorption from chromatographic materials canLC-MS and CESI-MS characterizations of proteins performed

plague LC-MS analyses of intact proteins, surface coatings tousing a bottom-up proteomics (Level 3 peptide mapping)

suppress protein-wall interactions in CE have been applied withstrategy are common and provide a wealth of information aboutsuccess. For example, a positively-charged polymer likesequence and post-translational modifications (i.e. quality

polyethyleneimine (PEI) can be covalently attached to a

attributes), crucial information about intact protein forms can becapillary’s inner wall to eliminate protein-wall interactions at low

lost during the digestion process. Top-down proteomics andpH and facilitate highly efficient CE separations of proteins for

Characterization Levels 1 (intact mAb) and 2 (reduced mAb) inMS analysis. In this case, the cationic polyethyleneimine (PEI)

the biopharmaceutical industry provide critical information about

coating is attached to the capillary wall through covalent siloxane

intact proteins using MS, but benefit from or require a separation

bonds similar to those commonly used for attachment of LC

step prior to electrospray ionization (ESI)-MS due to sample

stationary phases to chromatographic resins, providing a stablecomplexity. Capillary Electrospray Ionization (CESI) is thecoating for MS analysis.

integration of capillary electrophoresis (CE) and electrosprayionization (ESI) into a single process within the same device.
CESI-MS operates at low nL/min flow rates offering several
advantages including increased ionization efficiency and areduction in ion suppression.

This technical note describes the necessary steps to covalently
attach PEI to the surface of a bare fused-silica CESI cartridge
capillary. Additionally, a suitability test using protein standards asa means to evaluate capillary performance for the separation of
intact protein will be described.

Thus, CESI-MS is ideally suited for top-down proteomics andCharacterization Levels 1 and 2 of mAbs.

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Go to Method Menu > Instrument Set up. The Initial

Materials and Methods

Conditions Tab (Fig. 1), shows a set up that is common toThe coating procedure requires three steps: (1) pre-conditioning

all methods. Figures 2-6 and 8 show the time program forof the capillary surface, (2) coating the capillary surface with PEI

each method.

, and (3) purge unreacted polymer using a post-coating wash. All
three steps are conveniently performed on the CESI 8000 using
provided instrument methods.

Reagents needed:
Reagent

Catalog no.

VendorMethanol 0.2 mm filtered,Fisher Scientific Optima

A454-1

FisherScientific

Ntrimethoxysilylpropyl)polyehyleneimine (PEI) 50% inisopropanol

SSP-060-100gm

Gelest Inc.

5 M Sodium Chloride – 500 mL

59222C-500 mL

Sigma AldrichProtein Test Mix

477436

Sciex

Methanol (MeOH) anhydrous
99.8%322415-100 mL

Sigma Aldrich

Figure 1. Initial Conditions tab

Figure 2. Pre-conditioning method

Supplies needed from third party vendors:
Figure 3. Overnight coating method
Materials
15 and 50 mL conical bottom tubes
Assorted pipettes and corresponding tips

Figure 4. Post-coating purge method

Microcentrifuge Vials
3 mL syringe with needle
0.5 mL centrifuge vials

Figure 5. Capillary conditioning method

Supplies needed from Sciex:
Material

Catalog no.

CESI 8000 vials

B11648CESI 8000 caps

B24699OptiMS Silica Surface Cartridge

B07367

Figure 6. Capillary performance separation method
Note: To avoid premature capillary failure, it is strongly

Instrument methods settings usedrecommended that every voltage separation step is followed by avoltage ramp down with a 5 psi forward pressure. To set up thisThe instrument parameters used to run the various

step properly ensure the separation step window is set up asmethods are described below. These methods must be

shown in Fig. 7.

created using the CESI 8000 control software.
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Step 2. Prepare 100 mM acetic acid (HAc) solution by diluting
the glacial acetic acid. Dispense 287 L of HAc into a 50 mL
volumetric flask and complete to 50 mL with DDI water. This canbe kept refrigerated for up to 6 months.
Step 3. Adjust the pH of the HAc solution with the NH4Acsolution to pH 3.1. This can be kept refrigerated for up to 6months.
Preparation of system suitability stock solution:
The system suitability sample is a mixture of 3 proteins(Cytochrome C, Lysozyme, Ribonuclease A) at a total protein

Figure 7. Separate step settings used in the ramp down of theconcentration of 3 mg/mL. The sample is provided lyophilized as

separation method.

a combination of 1 mg of each protein. Add 1.0 mL of DDI water
to the vial and mix well to reconstitute the system suitability
sample. This can be kept refrigerated for up to 1 month.
Preparation of system suitability sample (for injection):
Aliquot 10 L of system suitability protein mix into aFigure 8. Capillary storage/shutdown method

microcentrifuge vial and add 10 L of 100 mM NH4Ac pH 3.1 and80 L of DDI water and finger flick for proper mixing. ThisReagents and Sample Preparation
1M Sodium Chloride (NaCl) - Positively coated capillary

mixture results in 0.3 g/L of protein mix at a total volume of100 L in 10 mM NH4Ac.

cleaning solution
High concentration sodium chloride is used to aid in the cleaning

Description of the Coating Processof the coated surface of anionic species present in the sample
that may have adsorbed onto the surface. The mechanism for

The coating process is best performed overnight since thethe removal of anionic contaminants from the cationic PEI

coating reaction is complete only after 12 hrs. Therefore it issurface is similar to anion exchange chromatography. The NaClstrongly recommended to begin the pre-conditioning method atis removed from the capillary by subsequent water and BGE

least an hour before the end of your day. The post coating purgerinses. The NaCl solution can be prepared by direct dilution ofwash can be started the next day.

the 5M stock solution. Dispense 2 mL of the 5M stock solution
into a suitable flask and add 8 mL of DDI water. This can be kept
refrigerated for up to 6 months.
Preparation of the BGE solution:
Step 1. Prepare 100 mM ammonium acetate (NH4Ac) solution bydiluting the stock solution of 7.5 M NH4Ac. Dispense 666 L of
7.5 M ammonium into a 50 mL volumetric flask and add DDI
water for a final volume of 50 mL. This can be kept refrigeratedfor up to 6 months.

Step #1: Capillary pre-conditioning
In preparation for the coating, the silica surface must be cleanedof any impurities that may be present on the surface. The
capillary surface preconditioning is comprised of a method inwhich the capillary undergoes a series of rinses with NaOH andHCl followed by water and MeOH. These steps serve to open
siloxane groups and to deprotonate (during rinses with NaOH),
protonate (during rinses with HCl), and wash (rinse with waterand MeOH).

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For this step, 1.5 mL of 0.1 N NaOH, 0.1 N HCl, DDI water andPreparation of the coating solution requires a clean working area

MeOH are dispensed in the CESI vials and capped. The vials

free of dust. If a gas line with dry Nitrogen, Helium, or Argon ismust be placed in the appropriate position as described in Fig. 9.

available, have clean stainless steel tubing on hand to draw themethanol from the bottle. If gas lines are not available, set aside
a 3 mL syringe with clean needle.
Draw 1.5 mL of methanol by piercing the septum of theanhydrous methanol bottle and dispense it into a 15 mL conicalbottom tube and cap immediately. Using the stainless steel
needle, fill the head space of the anhydrous methanol bottle withdry, inert gas (N, He, or Ar). If inert gas is not available, cap thebottle immediately and place it into a desiccator for storage. It isFigure 9. Reagent vials layout for capillary pre-conditioning

important to perform this step to keep the MeOH anhydrous.

procedure
Aliquot 300 L of the PEI solution and dispense into the tube
Prepare two 50 mL conical bottom tubes each containing 4 mL ofcontaining the anhydrous MeOH. The PEI solution is viscous andDDI water and 4 mL of filtered MeOH. Set the vial containing

will deposit at the bottom of the vial and it may also adhere to theMeOH aside. Place the vial containing DDI water into the holster

pipette wall. Pipette up and down repeatedly to ensure theon the right side panel of the CESI 8000 by screwing it in until it

polymer from the tip is dissolved into the methanol. Cap the tubeis snug. Remove the protective sleeve from the sprayer housingand vortex vigorously until all of the PEI is dissolved into theand rest it in the holster so the sprayer tip is immersed in the DDI

MeOH. This may take several minutes.

water. Be careful not to expose the tip.
TIP: If the PEI solution is kept refrigerated, it will be extremely
Important Note: Throughout the entire coating procedure, theviscous and drawing with a pipette tip will be very difficult. Simply

porous sprayer must be immersed in either DDI water or MeOH

cut the tip of the pipette tip (about 1 cm) with a clean razor blade

depending on the step. During step 1 or capillary preprior to drawing the polymer solution.

conditioning, the sprayer end must be immersed in 4 mL of DDI
water for the first 50 minutes of the method. Once the rinse withDDI water has completed, and the MeOH rinse has begun,
remove the 50 mL tube containing DDI water from the holster,
carefully and gently dab the sprayer housing dry with a tissue
paper, and install the 50 mL tube containing MeOH into theholster. There is no need to stop or pause the method while

Important note: The PEI solution must be prepared fresh
every time and no more than 15 minutes prior to coating.
Dispense 1.5 mL of the coating solution into a CESI vial. Cap thevial and place it in the A2 position of the buffer inlet tray into theCESI 8000 instrument. Discard the remaining PEI solution.

switching tubes. Under no circumstances should you touch

Dispense 4 mL of filtered MeOH into a new 50 mL conicalthe sprayer tip.

bottom tube and install this tube in the holster. It is important for
the end of the sprayer to be resting in fresh, clean MeOH.

Step #2: Capillary coatingAfter the capillary surface has been properly conditioned andStart the coating method shown in Fig.3.

activated, it is ready to be covalently coated with PEI. Thismethod will rinse the capillary for only 30 minutes; however theFollowing the overnight incubation period, the capillary is

polymer solution should remain inside the capillary overnight tocovalently coated with the cationic polymer and any unreacted

complete the reaction.

PEI must be removed. To avoid unwanted carry-over of
unreacted PEI into separation methods, it is strongly
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recommended to clean up the instrument interface before

The instrument should now be cleaned of residual PEI and isproceeding with the post coating purge method.

ready for the post-coating purge wash step.

Steps for instrument clean up:

The post-coating purge procedure consists of a series of1.

Remove all previously used vials and discard

methanol rinses which allows for the removal of any unreacted

appropriately. Place a fresh 50 mL tube of methanol

PEI from inside the capillary.

into the holster to keep the porous tip immersed at all

2.

3.

times to avoid it from drying out or plugging.

Dispense 1.5 mL of filtered MeOH into 3 CESI vials and cap

Carefully remove the CESI inlet vial ejector by pulling it

them. Dispense 4 mL of filtered methanol into a 50 mL conicalstraight downward to expose the capillary and electrodebottom tube. Install the tube on the holster of the CESI 8000 andfor cleaning.

place the newly prepared CESI vials into the buffer trays as

Using a lab tissue soaked with methanol, gently wipeshown in the Fig. 11.

the inlet capillary and inlet electrode clean of PEI.
4.

Remove the cartridge from the interface block and rest
it on the transport rails (Fig 10). Clean the underside of
the inlet interface block.

Important Note: ALWAYS remove the cartridge BEFORE
removing the electrode. Failure to do so will break theseparation capillary.
5.

Remove the CESI electrode using the electroderemoval tool.

6.

Place both electrode and vial ejector in a beaker

Figure 11. Reagent vials positioning for capillary post purge

containing methanol (MeOH does not need to be highprocedure

quality grade). Sonicate the beaker for 10 minutes.
7.

8.

Dry the electrode and ejector and re-install them into

This step completes the coating and the capillary is now ready tothe CESI 8000.

be conditioned and then tested on the CESI 8000.

Re-install capillary cartridge.

Notes:


If the capillary isn’t used right away, it can be stored
using the storage/shutdown method provided in Fig. 8.
When not in use, the PEI capillary must be stored dry.
Always use vacuum to dry the separation capillary as
shown in the storage/shutdown method in Fig. 8.

Conditioning of the positively-coated capillary
To obtain the best performance from the PEI capillary, it isnecessary to condition prior to running a test sample. The
conditioning is a simple procedure, consisting of applying -20 kV
for 20 min. The optimum electrospray voltage is also determinedat this point. For this procedure both separation and conductive
liquid capillaries must be filled with BGE 100 mM NH4Ac pH 3.1.
Preparation of the BGE is detailed in a later section.

Figure 10. Rest the cartridge over the rails while keeping theporous tip submerged in methanol.

p5With the protective sleeve removed from the sprayer housing,Note: Confirm your ESI spray conditions in the last conditioning

install the sprayer into the adapter on the MS source.. Dispense

step with the information in the User Manual as it may be

1.5 mL of the BGE, 1M NaCl, and DDI water into their respective

dependent on your MS instrument model.

CESI 8000 buffer vials and cap them properly. Place the vials in
the proper positions as indicated in Fig.12.

Testing the PEI-coated capillary
Dispense 100 L of the system suitability sample into a samplevial. Place the sample vial into the CESI buffer vial and cap it.
Place the CESI vial into position A1 of the inlet sample tray.

The separation method used to test the performed is shown inFig. 5.
Dispense 1.5 mL of the BGE, 1M NaCl, and DDI water into theCESI 8000 buffer vials and cap them properly. Place the vials inthe proper buffer tray positions as indicated in Fig.12.

Figure 12. Layout of reagent vials required to condition and test
performance of PEI-coated capillary

Important Notes:
Use the capillary conditioning method as shown in Fig. 4. Uponcharged, the electroosmotic flow (EOF) generated whencompletion of the conditioning method, align the tip of theapplying voltage in normal polarity would flow insprayer with your specific mass spectrometer as described in theopposite direction relative to the MS. For this reason theUser Manual or Application Guide. Using the direct control

separation method is done under reverse polarity soscreen in 32 Karat, apply 20 kV and reverse polarity with a ramp

EOF now flows towards the MS. Reverse polarity is

time of 1 min for a duration of 60 minutes (Fig. 13). Program theprogrammed into the CESI method provided for PEI-MS-ESI voltage to start with the 0 kV and increase slowly until
spray is observed. Optimize the position of the tip and the sprayvoltage until a minimum ESI voltage needed for stable spray isBecause the surface of this capillary is positively

based separations.
The separation step in the method (line 9 of the method
shown in Fig.6) is for a voltage ramp down. Please

determined. Allow the capillary to spray for 20 min to complete

ensure this step is performed with 5 psi forward

conditioning.

pressure. The voltage ramp down is programmed into
all of the CESI separation methods.
To ensure a stable spray throughout the separation,
always add 100 V to the minimum ESI voltage foundduring the capillary conditioning procedure for the MS
acquisition method.

Figure 13. Direct Control settings for PEI capillary coatingconditioningp6

Fig. 14 shows the expected base peak electropherogram andcorresponding spectra.

21

Peak 1

3

Peak 2

Peak 3Figure 15. Vial layout used in the shutdown method

Conclusion
Figure 14. Base peak electropherogram and spectra expected

This technical note describes a simple method for coating barefor the three protein mix separated using a PEI-coated capillary.

fused-silica OptiMS capillaries for intact protein and polypeptide

Peak 1 is Lysozyme, peak 2 is Ribonuclease A and peak 3 isseparation applications. Use of the PEI coating creates a robust

Cytochrome C.

and reproducible means to eliminate protein adsorption onto thecapillary surface when performing CESI-MS. The covalent nature

Storage of the positively-coated capillaryof the siloxane-linkage allows for very stable PEI coating at

Capillary storage following use requires running the shutdown

acidic pH. The coating can be stripped off the surface andmethod as shown in Figure 8. Dispense 1.5 mL of DDI water andrecoated by using the same procedure described in this note.

filtered MeOH into the CESI 8000 vials and cap them. Place theWhen used in CESI-MS, the PEI coating procedure provides avials in the buffer tray positions shown in Fig.15.

powerful tool for analysis of intact or complex protein cohorts
associated with biotherapeutic characterization or top down

Note: It is not recommended to set up the shutdown method

proteomics.

following a long batch run as the methanol may evaporate before
completion. To circumvent this, add or fill the methanol vial
immediately before the shutdown method is run.

© 2015 AB Sciex. For Research Use Only. Not for use in diagnostic procedures. AB Sciex is doing business as SCIEX.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.
Document number: RUO-MKT-18-2325-A

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