Optimized digestion procedure and characterization formonoclonal antibodies and proteins by CESI-MS
Featuring CESI 8000 Plus and TripleTOF® 6600+ LC-MS/MS System
Zuzana Demianova1, Anna Lou1 and Sahana Mollah2
1 SCIEX, Brea, CA; 2 SCIEX, Redwood City, CA
Peptide mapping by liquid chromatography-mass spectrometry(LC-MS) is a widely utilized technique to characterize
monoclonal antibodies (mAb) and protein therapeutics. It
employs a bottom-up approach where the mAb of interest isdigested with an enzyme, such as trypsin and the resulting
peptides are analyzed by tandem mass spectrometry (MS/MS).
Reverse-phase C18 columns are a primary choice for peptide
mass mapping of mAb, but characterization of post-translation
modifications (PTMs), such as various type of glycosylations andidentification of very large hydrophobic1, and small hydrophilic
peptides can be a challenge.2 Capillary electrophoresis-mass
spectrometry (CESI-MS) separates analytes by by their ionic
mobility related to the charge/mass ratio that makes it anorthogonal technique to peptide mass mapping by LC-MS.
CESI-MS offers peptide identification with high-sequence
coverage (Figure 1) and characterization of multiple PTMs in asingle analysis2,3,4. CESI-MS operates at nano flow range (~ 40nL/min), increasing ionization efficiency and reducing ion
suppression.
Using a preconcentration technique such as transientisotachophoresis and capillary zone electrophoresis (t-ITP-CZE)
enables greater sensitivity due to high sample loading. 4 In
addition it increases the assay sensitivity and separation
resolution of glycopeptides.2,4

CESI 8000 Plus and TripleTOF® 6600+ System

Further t-ITP-CZE increases the assay sensitivity andseparation resolution of glycopeptides.2,5,6
In this technical note, we provide a monoclonal antibody andprotein digestion procedure specific for CESI-MS application,
since it is very important to obtain a high sequence coverage ofmAb and biotherapeutic proteins.
Furthermore, we summarized a good practice for peptide
analysis by CESI-MS.

Key Features of CESI 8000 Plus coupled to
TripleTOF® 6600+ System

Figure 1. High sequence coverage of light and heavy chains of
Adalimumab using the sample prep and optimized conditions
provided in this technical note. Green color: high confidence;
Orange color: middle confidence•High sequence coverage of monoclonal antibody or protein
based biotherapeutics•

Electrophoretic separation of various glycopeptides•

Minimum sample used (nanoliters) to obtain high sensitivity
providing identification of low level PTMs.

•

MS/MS confirmation of short, very large hydrophobic, andsmall hydrophilic peptides often missed in LC-MS

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Methods
Equipment
Temperature adjustable water bath or thermoshaker (37 °C to 50°C), speedVac. dryer, CESI 8000 system, OptiMS BFS Capillary(SCIEX, B07362), CESI adapter (P/N B07366), SCIEX mass
spectrometer with NanoSpray® Ion Source III (TripleTOF® 6600+
System), CESI vials (SCIEX, B11648), CESI caps (SCIEX,
B24699), PCR microvials (SCIEX, 144709), Nanovials (SCIEX,
5043467).

inverting the flask 3 times. It can be store at 2 °C to 8 °C up to 24
months.
BGE (10% (v/v) HAc): Add 2.0 mL of HAc into the 18 mL of DDI
water and mix it well. Sonicate solution at least 15 min prior touse. IMPORTANT: This solution must be prepared fresh everyday.
Tips for optimal results:
1.

Avoid using non-volatile salts because it might resultin plugged sprayer tips.

2.

Avoid using extremely low conductivity buffers
because they mighty result in sprayer tip breakages andside leaks.

3.

Avoid using BGE’s with high ionic strength (e.g.
10% Formic acid) to keep the separation current lower
than 5 µA as it might cause permanent damage to theseparation capillary and its coating.

Reagents
RapiGest SF (Waters, 186008090), Trypsin Gold (Promega,
V5280), Ammonium bicarbonate (AmBic, Millipore Sigma,
A6141), Dithiothreitol (DTT, Promega, V3155), Iodoacetamide(IAA, Millipore Sigma, I1149), Acetic acid, glacial (HAc, Millipore
Sigma, A6283), Formic acid (MS-grade, Fisher-Scientific, A11750), 7.5 M Ammonia Acetate (Millipore Sigma, A2706), 5 M
Sodium Chloride (NaCl, Millipore Sigma, S6546), 1 mol/L (1 N)
Hydrochloric acid (HCl, Millipore Sigma, 1090571000), Methanol(MS-grade, Millipore Sigma, 900688), tuning solution part of the
TripleTOF 6600+ system installation kit (SCIEX, 4456736).
Reagent preparation
500 mM dithiothreitol (DTT): weight 77 mg in a microcentrifuge
tube and add 1 mL of DDI water.
500 mM iodoacetamide (IAA): weight 92 mg IAA in a
microcentrifuge tube and add 1 mL of DDI water. Solution should
be made fresh each time and kept in dark.
500 mM ammonium bicarbonate (AmBic): weight 3.95 g into a
volumetric flask and adjust with DDI water to 100 mL.
Enzyme preparation (Gold Trypsin): reconstitute Trypsin with 50
mM acetic acid to a concentration of 1 μg/μL.
20% acetic acid (HAc): add 10 ml of HAc into 40 mL of DDI
water.
0.1 M NaOH add 1 mL of NaOH to 49 mL of DDI water.
0.1 M HCI add 5 mL of HCI to 45 mL of DDI water.
Leading and background electrolytes (LE and BGE)
LE (200 mM ammonium acetate pH 4): Make 50 mL 400 mM
ammonium acetate (AmAc) solution (Add 2.7 mL of 7.5 M AmAc
into 47.3 mL of DDI water) and adjust the pH of the solution to
4.0 by adding aliquots of 20% HAc freshly prepared. Adjust thevolume of final solution to 100 mL and mix the solution by

Sample preparation prior digestionmAb digestion buffer: Add 200 μL of 500 mM ABC into RapiGest
SF vial for a final concentration of 0.5 μg/mL
mAb sample: Add 100 μg of selected mAb into a 1.5 mL
microcentrifuge tube, add 20 μL of mAb digestion buffer, thenadd 60 μL DDI water to make a final volume of 80 μL.
TIP: The surfactant RapiGest SF breaks-down into noninterfering and non-ion suppressing products by acidifying final
sample mix.
TIP: Therapeutic proteins or mAbs with higher hydrophobic
characteristic, might require a higher amount of the RapiGest
SF.3
Sample digestionmAb reduction and alkylation
Step 1. Add 4 μL of 0.5 M DTT to mAb sample for a final
concentration of 10 mM DTT and mix the sample mixture
well by quick vortexing
Step 2. Incubate the sample at approx.+56 °C for 45 minutes
Step 3. Cool to room temperature for 10 minutes
Step 4. Add 4 μL of 0.5 M IAA into the sample mixture andincubate the sample in dark for 30 minutes
Step 5. To quench the alkylation reaction, add 2 μL of 0.5 M DTT
into the sample mixturep2

Monoclonal antibody and protein digestion

Sample preparation prior CESI-MS

Step 1. Prior to enzyme digestion, add 5 μL of DDI water for a
final volume of 95 μL in sample mixture

Monoclonal antibody used in this case was adalimumab.
Digested mAb sample was diluted in a 1:2 (v:v) ratio with 200
mM ammonium acetate at pH 4.

Step 2. Add 5 μL of reconstituted trypsin for a final 1:20
enzyme-to-protein sample ratio
Step 3. Finger flick the sample tube to mix
Step 4. Incubate the sample mixture at the approx.. +37ºC for 2
hours
Step 5. After digestion, quick spin the sample tube for 10
seconds
Step 6. Add 1 M formic acid to the sample mixture for a final
concentration of 0.5% (11 μL of 1 M formic acid to 100 μL
sample reaction mixture)
IMPORTANT: Other proteases can be used for mAb or protein
digestion; the protein/enzyme ratio and/or digestion time willneed to be adjusted adjust depending on enzyme manufacturer
instructions.

It is generally recommended to have final LE concentration of
100 mM ammonium acetate pH4 in the sample. In a few cases,
The higher concentration of LE might help focusing the sample
especially when lower separation voltage is used.
To adjust sprayer position a pI marker (pI 9.5, 5 µL plus 95 µL of
BGE) was infused. Further instructions can be found in BFS
CESI manual, chapter 5.6
IMPORTANT: Every sample that is mixed with LE needs to be
centrifuged for at least 5 min at 14 000 rpm, to avoid cartridge
clogging by precipitant.
CESI
For the peptide mapping separation, we used a Bare-Fused
Silica Surface OptiMS (BFS) Cartridge.

Tips for digested sample storage:
OptiMS cartridge conditioning:••

For digest in solution: Aliquot 20 μL of sample
mixture, add 40 μL of LE buffer pH 4.0. This sample
may be stored at 2-8 oC for one week. Centrifuge andremove pellet prior to analysis as described in criticalstep below.
For lyophilized digest: Dry 100 μg of digest using acentrifugal vacuum concentrator. Store the digest at -15
to -30 oC for approx. half a year..

Table 1. CESI cartridge conditioning

Event

Time Pressure Direction(min) (psi)

1 Rinse10

100

2 Rinse3100

3 Rinse10

100

4 Rinse3

100

Solution/Note

ForwardMethanol/emitter dip inmethanol

ReverseMethanol emitter dip inmethanolForward

DDI water/emitter dip in DDI
water

ReverseDDI water/emitter dip in DDI
water

Forward

0.1 M NaOH/emitter dip in
DDI water

Forward

0.1 M HCI/emitter dip in DDI
water

Prior to use, the BFS cartridge was conditioned following aseries of rinses as described in Table 1.
Adjusting the sprayer position:
To adjust sprayer position a pI marker (pI 9.5, 5 µL plus 95 µL of
BGE) was used. Sprayer position should be at least 2-3 mm fromthe orifice to avoid tip clogging due to temperature and voltage
applied.Further instructions can be found in BFS CESI manual,
chapter 5.4
Table 2. CESI separation conditions used for the peptide mapping

Time Pressure Voltage Direction(kV)(min) (psi)Solution/Note

1 Rinse2.5

100

Forward

0.1 M NaOH

2 Rinse2.5

100

Forward

0.1 M HCI

3 Rinse4

100

Forward

DDI water

4 Rinse3

75

Reverse

10% Acetic acid

5 Rinse4

100

Forward

10% Acetic acid

6 Injection

60 s

5

Forward

10% Acetic acid

7 Wait00Forward

DDI water

8 Injection

25 s

0.5

Forward

10% Acetic acidEvent

5 Rinse10100

6 Rinse10

100

7 Rinse10

100

Forward BGE/emitter dip in DDI water

9 Separation

40

0

20

Forward

10% Acetic acid

8 Rinse3

100

Reverse BGE emitter dip in DDI water

10Separation

5

0

1

Forward

5min ramp down

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The starting ion spray voltage and sprayer position needs to be
optimized per each cartridge and can vary from 900 to 1 800 V.
In
Table 3. CESI overnight and over weekend storage

Event

Time Pressure(min) (psi)

Direction

Solution/Note

1 Rinse5

100

Forward

0.1 M NaOH

2 Rinse5

100

Forward

0.1 M HCI

3 Rinse5

100

ReverseDDI water

4 Rinse999

5

Forward

DDI water

addition, Sprayer position should be at least 2 mm from theorifice to avoid tip clogging due to temperature and voltage
applied.
Table 4. CESI long time storage

Event

Time Pressure Direction(min) (psi)Solution/Note

1 Rinse5

100

Forward

0.1 M NaOH

2 Rinse5

100

Forward

0.1 M HCI

3 Rinse5

100

ReverseDDI water

4 Rinse5

100

Forward

DDI water

5 Vacuum

10100

ForwardDry (Storage)

6 Vacuum

10100

ReverseDry (Storage)

CESI-MS separation conditions:
The Adalimumab digest sample in LE was injectedhydrodynamically for 60 s at five psi, resulting in an injection
volume of approx. 44 nL, corresponding to a sample load of 39
ng. Table 2 describes the separation conditions for the peptide
mapping method.
TIP: It is recommended to lower the separation voltage to allow
for better separation of glycopeptide species that are difficult toresolve (Figure 2). Example, for separation voltage of 15 kV, theseparation analysis time is 50 minutes.2
IMPORTANT: Generally, it is not recommended to apply aseparation voltage across the BFS cartridge that exceeds 20 kV.
After use and to increase capillary life, it is recommended to use
the method described in table 3. This method allows over 16hours of a gentle and continuous flow of DDI water through theseparation capillary keeping it from clogging while on board the
instrument. If the capillary cartridge won’t be used for longer thanovernight period, it is recommended to be stored dry by following
the steps in table 4.

Mass spectrometry
The TripleTOF® 6600+ System was coupled with the
NanoSpray® Ion Source III with CESI adapter and controlled byAnalyst® Software 1.8.1 . The peptide mapping of adalimumab
was performed using data dependent acquisition (DDA) with 10
MS/MS cycles (150 and 50 ms accumulation times for MS and
MS/MS, respectively). The acquisition mass range was from 350
to 2250 m/z with cycle time of 0.8 s. The precursors with charges
from +2 to +5 and intensity above 100 cps were triggered for
MS/MS fragmentation selected precursors were fragmented
using a rolling collision energy (CE) with spread of ± 5 V. Source
parameters are listed in the Table 5. The starting ion sprayvoltage and sprayer position needs to be optimize per eachcartridge.
Separation performance of the cartridge and instrument
calibration can be checked using standards. To set up the
autocalibration follow the guidelines in CESI manual, chapter 5. 4
IMPORTANT: A steady flow of ions through the sheathlessinterface is critical for a stable spray and reproducible
separation, which can be accomplished by using volatile
reagents such as acetic acid.
Table 5. Summary of mass spectrometry parameters.
Parameter

Value

Parameter

Value

MS mass range 350-2250 m/z MS/MS mass range 150-2250 m/z
MS accumulation 150 ms
time

MS/MS
accumulation time

50 ms

Curtain gas:

5 psi

Source temperature: 50 °C

Polarity:+

Ion spray voltage:

900-1800 V

Data processing: ProteinPilotTM and BPV Flex Software wasused for mAb characterization and SCIEX OS-Q Software wereused for peptide mapping analysis.

Results and discussion
CESI-MS peptide mapping
CESI-MS is an effective and complementary technology to LCMS for the characterization of mAbs at the peptide level with ahigh sequence coverage. In the example case, we confidently
map at least 99% of adalimumab sequence (Figure1).
Additionally, trypsin digestion protocol was tested on
cetuximab3, transtuzumab4,7, bevacizumab and IgGs (data notshown, for detailed study of cetuximab and transtuzumab checkreference 3, 4 and 7). Due to high sequence coverage, micro-

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Figure 2. Characterization of adalimumab by CESI-MS. Left panel: Total ion chromatogram (TIC) of adalimumab digest; Right panel: Extracted ion
chromatograms (XICs) of low abundant high mannose N-glycans on aspartic acid 301 of EEQYNSTYR peptide. Further information can be found incomprehensive analysis of low abundant mannose glycopeptides in peptide mapping of adalimumab, RUO-MKT-02-11834-A.2

sequence changes between different biosimilars could bestudied (data not shown, for detailed study check reference 7).8

substitution as well as post-translation modifications that are
critical for a biotherapeutic’s safety and efficacy.

Post translation modifications

CESI-MS peptide mass mapping is a valuable orthogonaltechnology that provides additional sequence coverage

While performing peptide mass mapping of adalimumab various
glycopeptides were confirmed (data and further information arelisted in the technical note. Comprehensive analysis of low
abundant mannose glycopeptides in peptide mass mapping ofadalimumab)2, as example high mannose are shown in Figure 2.
While glycans are important post-translational modifications thatare expected on adalimumab, there are also other PTMs which a
can affect the safety, efficacy and even solubility of thebiotherapeutic.

compared to LC-MS. In this study, we presented peptide
mapping of adalimumab with >99% sequence coverage from~40 ng sample.

Figure 3, illustrates example of the separation between
unmodified vs. deamidated and oxidized form of three different
peptides of adalimumab. The deamidated peptide
FNWYDGVEVHNAK and YNRAPYTFGQGTK were determined
to be 3.2% and 2.9% of the total amount of peptide in thesample, respectively. The peptide NSLYLQMNSLR contains
8.6% of oxidized from total amount of peptide. Deamidation andoxidation are the most common PTMs occurring on therapeuticproteins produced using recombinant DNA technology.

Conclusions
Peptide mass mapping remains a key analytical methodology tocharacterize biotherapeutics from drug discovery throughproduction and it provides the ability to assign specific locationsof product attributes. It allows monitoring of amino acid

Figure 3. Deamidation of aspartic acids and oxidation ofmethionine in Adalimumab. Peptide FNWYDGVEVHNAK and
YNRAPYTFGQGTK were determined to be 3.2% and 2.9% of the total
amount of peptide in the sample, respectively. From total amount of
peptide NSLYLQMNSLR 8.6% contains oxidation of methionine.

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Furthermore, critical quality attributes, such as glycosylation,
deamidation and oxidation of peptides, can be characterizeduring the peptide mapping analysis without additional
measurements.3,4,8

electrophoresis-tandem mass spectrometry. Anal
Chem. 2014;86(18):9074‐9081.

References
1.

Improving the detection limits for highly basic
neuropeptides using CESI-MS, RUO-MKT-02-7150-A,
https://sciex.com/Documents/tech%20notes/technology/
CESI/Neuropeptide-detection-CESI-MS.pdf

2.

Comprehensive analysis of low abundant mannose
glycopeptides in peptide mapping of adalimumab, RUOMKT-02-11834-A

3.

Gahoual R, Biacchi M, Chicher J, et al. Monoclonal
antibodies biosimilarity assessment using transientisotachophoresis capillary zone electrophoresis-tandem
mass spectrometry. MAbs. 2014;6(6):1464‐1473.

4.

Gahoual R, Burr A, Busnel JM, et al. Rapid and multilevel characterization of trastuzumab using sheathlesscapillary electrophoresis-tandem mass
spectrometry. MAbs. 2013;5(3):479‐490.

5.

Yu, Ying-Qing, et. al. A complete peptide mapping of
membrane proteins: a novel surfactant aiding the
enzymatic digestion of bacteriorhodopsin. Rapid Commin Mass Spectrom. Letter to the Editor. 2004; 18; 711715.

6.

CESI 8000 High Performance Separation System, Usermanual, RUO-IDV-05-3897-C March 2020
https://sciex.com/Documents/manuals/cesi8000_uManu
al_B11949AD.pdf

7.

Lew, C., Gallegos-Perez, J.L., Fonslow, B., Lies, M. andGuttman, A., 2015. Rapid level-3 characterization oftherapeutic antibodies by capillary electrophoresis
electrospray ionization mass spectrometry. Journal ofchromatographic science, 53(3), pp.443-449.

8.

Gahoual R, Busnel JM, Beck A, François YN, LeizeWagner E. Full antibody primary structure andmicrovariant characterization in a single injection usingtransient isotachophoresis and sheathless capillary

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