Drug discovery and development

Preparation Scale-Up of Complex Biological Samples
for Deep N-glycomic Analysis by CE-LIF and CESI-MS
Andras Guttman1,2 and Marton Szigeti2
1 SCIEX, Brea, CA, US
2 Horváth Csaba Memorial Laboratory of Bioseparation Sciences, University of Debrecen, Hungary

Introduction
Complex biological samples, including bioprocessing
specimens in the pharmacological sector, contain proteins in awide concentration range of pg/mL to mg/mL levels.1 Most ofthese proteins are post-translationally modified, including theattached carbohydrates, which allegedly influence their activity,
physicochemical properties and effector functions.2 Therefore,
deciphering the glycan moieties of such proteins is important inboth the biomedical and biopharmaceutical arenas. For deep
N-glycomic analysis of biological and bioprocessing samples
using high-performance glycoanalytical tools—such as capillaryelectrophoresis with laser-induced fluorescence (CE-LIF)
detection or its hyphenated format, capillary electrophoresis withelectrospray ionization mass spectrometry (CESI-MS)—efficientand precipitation-free sample preparation methods are needed

In this technical note, an efficient and scalable sample
preparation workflow is introduced to accommodate deep
N-glycomics analysis of complex biological samples containing
up to milligrams of proteins. The protocol includes the captureof glycoproteins by amine coated magnetic beads alongwith a novel temperature gradient denaturation process to
avoid precipitation. The workflow also helps to significantly
decrease the concentration of free low molecular mass
sugar contamination, minimizing their inhibition effect on
endoglycosidase digestion-based oligosaccharide removal.
Most importantly, application of the new sample preparation
scale-up workflow (Figure 1) is made possible to provide theappropriate amount of N-linked carbohydrates for deep glycomic
analysis by CE-LIF and CESI-MS, even in negative ionization
mode for the latter.

to provide the sufficient amount of analyte for high sensitivity.

Figure 1. Sample preparation scale-up workflow for deep N-glycomic analysis of complex biological samples by CE-LIF and CESI-MS.

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Drug discovery and development
Key features of the C100HT Biologics
Analyzer System• An efficient sample preparation scale-up workflow is
introduced for deep N-glycomics analysis of complex
biological samples and bioprocessing specimens• Protein precipitation is prevented by the novel temperature
gradient denaturing protocol

The supernatant was dried and labeled with 8-aminopyrene1,3,6-trisulfonic acid (APTS) utilizing a modified evaporative
labeling protocol of 1 h at 50 °C with closed lid vials and 1 h at
55 °C with open lid vials.3 The excess APTS dye was removed
by following the sample preparation and analysis protocol of
the Fast Glycan Labeling and Analysis Kit (SCIEX). The purified
samples were analyzed by CE-LIF and CESI-MS.

• The free sugar content in the complex biological samples
is significantly decreased, enabling improved endoglycosidase
digestion efficiency

Capillary Electrophoresis with Laser-Induced Fluorescence• The fluorophore labeling yield is enhanced by the modified
evaporative labeling protocollaser with 488 nm excitation and 520 nm emission wavelengths

Methods

and 20 cm, depending on the application. The respective electric

Chemicals: The Fast Glycan Labeling and Analysis Kit (SCIEX,

mode. The separation temperature in all analyses was 30 °C, andBrea, CA) included the tagging dye of 8-aminopyrene-1,3,6-

the samples were injected using a 5 s/3–5 psi water pre-injection

trisulfonic acid (APTS), HR-NCHO separation gel buffer and thefollowed by electrokinetic sample injection by 1–6 kV/1–3 s andpurification components. The amine functionalized magnetic

the injection of the bracketing standard by 1 kV/1 s. The 32

beads were from chemicell (Berlin, Germany). PBS, acetic acid,Karat™ Software 10.1 (SCIEX) was used for data acquisition.

ammonium acetate, isopropanol, sodium-cyanoborohydride(1 M in THF), tetrahydrofuran, HPLC grade water, acetonitrile,human IgG1 and human serum were from Sigma-Aldrich(St. Louis, MO). The PNGase F enzyme was from Asparia
Glycomics (San Sebastian, Spain).

(CE-LIF): A PA 800 Plus Pharmaceutical Analysis System(SCIEX) was used for all CE-LIF analysis utilizing a solid-stateand employing the HR-NCHO separation gel buffer. The effective
lengths of the 50 μm ID bare fused-silica capillaries were 50 cm
field strengths were 500 and 1000 V/cm, in reversed polarity

CESI-MS: All CESI-mass spectrometry tests used the CESI 8000
Plus High-Performance Separation - ESI Module, connected
to a QTRAP® 6500+ LC-MS/MS System (both from SCIEX)
through an OptiMS capillary cartridge (91 cm total length,
30 μm ID, 150 μm OD). All separations were done using

Temperature gradient denaturation: Glycoproteins from

10 mM of ammonium acetate, 0.1% acetic acid (pH 4.5), and50 μL of undiluted human serum (or 50 μL of 30 mg/mL of hIgG1

20% isopropanol containing MS friendly background electrolyte.

solution) were captured by 20 μL (50 mg/mL) of well-suspended

The applied voltage was 20 kV in reversed polarity mode withamine-functionalized magnetic beads mixed with 50 μL of 1 M

an additional 2 psi forward pressure at both the separation andsodium-cyanoborohydride (in THF) and 1.0 mL of 0.1x PBS.

the conductive capillaries. The cartridge temperature was set

After discarding the supernatant, the beads were washed withto 20 °C. Injection: 1 psi / 5 s water pre-injection followed by

1.0 mL of HPLC grade water and re-suspended in 20 μL of

10 kV / 20s sample introduction.

HPLC grade water. The captured proteins were denatured on
the beads by applying a temperature gradient of 5.0 °C/min, witha linear increase from 30 °C to 80 °C and an additional 5.0 min
isothermal incubation step at 80 °C in a PCR machine (Model
PTC-100, Marshall Scientific, Hampton, NH) in the presence ofImportant:• A separation current above 5 μA might cause permanent
damage to the separation capillary.

and Analysis Kit, SCIEX) with the addition of 12.5% glycerol.

• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

Endoglycosidase digestion: The PNGase F digestion

Mass Spectrometry: MS analysis used a QTRAP® 6500+

10 μL of premixed denaturation solution (Fast Glycan Labeling

was accomplished on the beads with the addition of 3 IUB(International Union of Biochemistry) units of enzyme(2.0 μL) followed by incubation at 50 °C for 1.0 h and finishedby precipitating the deglycosylated proteins onto the surfaceof the amine coated beads by 120 μL of ice-cold acetonitrile.

System in negative ionization mode. The ESI voltage was set
to -1,600 V for 1 min to stabilize the spray and to -1,400 V for
high-sensitivity detection. The orifice plate temperature was
80 °C with 5 psi curtain gas pressure for spray stabilization at the20.4 nL/min flow rate. The multiple reaction monitoring (MRM)
scan targeted the doubly charged species of APTS labeledp2

Drug discovery and development
glycan masses at 15 msec, and the data were evaluated andFigure 2 compares the CE-LIF electropherograms of the PNGasevisualized with PeakView® Software 2.2 (SCIEX).

F released, and APTS labeled human serum N-glycome using

Practicing traditional isothermal denaturation methods during theanalysis of protein glycosylation usually results in precipitation
when larger than several hundred micrograms of proteins areused, making downstream sample processing difficult or even
impossible. The recently introduced temperature gradient

2 and 5 μL starting material. With otherwise equal glucose
peak heights, the separation trace generated by the use of 5 μL
starting material unexpectedly resulted in lower peak intensities
for the N-glycome peaks than those that resulted from startingwith 2 μL human serum.

denaturation protocol successfully addressed this problem andFigure 3 shows the resulting average peak intensity changes.

allowed precipitation-free denaturation of up to 1.5 mg protein-

To understand this counterintuitive phenomenon, both the APTS

containing samples in a volume of 50 μL.

labeling and the PNGase F digestion processes were closely

4

scrutinized by the addition of extra glucose to the reaction
mixtures. Extra glucose was added prior to the APTS labelingreaction step (◦) and before the endoglycosidase digestion
reaction step (•). As one can observe, the APTS reaction kinetics
were not affected by the presence of the extra glucose, while
the PNGase F digestion reaction was significantly influenced,
losing most of its effectivity at the 1 mg/mL added glucose
level, probably due to the inhibitory effect of small sugars onreaction kinetics.4
Based on these observations, a novel sample preparation
scale-up workflow has been developed for deep N-glycomic
analysis by CE-LIF and CESI-MS, comprising the following steps.

1. Protein capture:
Figure 2. Comparative CE-LIF analysis of the human serum N-glycome
using 2 and 5 μL starting material. Conditions: HR-NCHO gel buffer in
50 cm effective (60 cm total) and 50 um ID bare fused-silica capillarycolumn; E=500 V/cm, t= 30 °C. Injection: water for 5 s at 5 psi, samples
for 3 s at 6 kV and bracketing standard 1 s at 1 kV.

- Add 50 μL of complex biological or bioprocessing sample
into a 1.5 mL Eppendorf tube.
- Add 50 μL of 1 M NaBH3CN (in THF) to the sample.
- Vortex the sample at maximum speed for 5 s.
- Wait for 1 min.
- Add 1.0 mL of 0.1x PBS solution.
- Add 20 μL of amine-functionalized magnetic beads(50 mg/mL, 1.0 μm diameter).
- Wait for 30 min at room temperature (RT).

2. Removal of the unbound proteins:- Remove the supernatant after pulling down the magnetic
beads by a magnetic stand.
- Reconstitute the beads in 500 μL of HPLC grade water.
- Vortex the sample at maximum speed for 10 s.
- Apply a quick (2–3 s) spin-down in a benchtop microfuge.
Figure 3. Effect of the extra glucose added to the reaction mixture on the
APTS labeling (◦) and PNGase F digestion (•) effectivity.

- Wait for 5 min at RT.
- Remove the supernatant while the vial is on themagnetic stand.
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Drug discovery and development
3. Temperature gradient denaturation:- Incubate the sample for 1 h at 50 °C with the lid closed.

- Prepare the denaturation solution by supplementing 70 μLof the Fast Glycan Labeling and Analysis Kit denaturation
solution with 10 μL of glycerol. (The Fast Glycan Labelingand Analysis Kit denaturation solution consists of: 50 μL of
D4 + 10 μLD1 + 10 μL of D3. Other similar purpose
denaturation solutions from other vendors may be usedwith the glycerol supplement.)- Incubate the sample for 1 h at 55 °C with the lid open (or until
the sample is completely dry).

- Add 20 μL of HPLC grade water to the beads.
- Apply a quick (2–3 s) spin-down in a benchtop microfuge toremove any sample from the wall of the Eppendorf vial.
- Add 10 μL of denaturing solution to the sample (do not touchthe beads with the pipette tip).

6. Purification and elution of the APTS
labeled glycans:- Reconstitute the dry sample with 20 μL of Fast Glycan
Labeling and Analysis Kit magnetic beads in water(concentrated from 400 μL of M1 process solvent by storage
solution replacement with water on a magnetic stand).
- Vortex the sample at maximum speed for 10 s (wait for
3 min for complete reconstitution after addition of thebeads, then vortex again).

- Vortex the sample at maximum speed for 10 s.

- Add 170 μL of acetonitrile to the sample (do not vortex
after this step).

- Apply a quick (2–3 s) spin-down in a benchtop microfuge.

- Wait for 1 min at RT.

- Denature the sample for 15 min using the following
temperature gradient: 30 °C to 80 °C using a 5 °C/minheating program, followed by 5.0 min incubation at 80 °C.

- Remove the supernatant after placing the vial on amagnetic stand.

4. PNGase F digestion on the bead:- Apply a quick (2–3 s) spin-down in a benchtop microfuge.
- Add 50 μL of HPLC grade water after the denaturation step.
- Add 2.0 μL of PNGase F enzyme.
- Vortex the sample at maximum speed for 5 s.
- Apply a quick (2–3 s) spin-down in a benchtop microfuge.
- Incubate the sample at 50 °C for 1.0 h.

5. Evaporative APTS labeling- Repeat the last 3 steps 3 more times using 20 μL of HPLC
grade water and 170 μL of acetonitrile per the Fast Glycan
Labeling and Analysis Kit user manual.
- Add 50 μL of HPLC grade water after the last supernatantremoval step.
- Vortex the sample at maximum speed for 10 s.
- Apply a quick (2–3 s) spin-down in a benchtop microfuge.
- Place the sample on the magnetic stand for at least 1 min.
- Transfer 45 μL of sample (avoid magnetic bead transfer)
into a new 200 μL PCR tube.

- Apply a quick (2–3 s) spin-down in a benchtop microfuge.

- Store the sample at 4 °C until analyzed.

- Add 120 μL of ice-cold acetonitrile.

- Use 5.0 μL of sample for up to 5 consecutive injections
in a CE nanovial for CESI-MS measurements.

- Keep the sample at -20 °C for 15 min.
- Centrifuge the sample at 10,700 x g for 5 min.

To demonstrate the advantages of this new and efficient
workflow, deep N-glycomic analysis of a human serum sample- Transfer the sample supernatant (200 μL) into a new 0.5 mL
Eppendorf vial.

was attempted. Figure 4 depicts the separation of the N-linked- Dry the sample in a SpeedVac for 60 min at 60 °C.

and CESI-MS (lower panel) using the amine-functionalized- Prepare the labeling stock solution of 9.0 μL of 20 mM APTS
(in 20% acetic acid), 1.0 μL of 1 M NaBH3CN (in THF) and10 μL of additional THF per sample.
- Add 20 μL of labeling solution to the dry sample (wash down
the sample from the Eppendorf wall with the labeling solution).

human serum oligosaccharides by CE-LIF (upper panel)magnetic bead-based protein capture with temperature gradientdenaturation-based sample preparation protocol. The use ofthis approach enabled a “precipitation-free” preparation of 50 μL
human serum sample, 25 times larger than that of the amountusually used to avoid precipitation (2 μL). Application of thisspecial protocol also removed the majority of the blood glucose- Vortex the sample at maximum speed for 10 s.

content from the sample, alleviating endoglycosidase enzyme- Apply spin-down for 10 s in a benchtop microfuge.

inhibition. All small peaks, otherwise buried in the baseline,p4

Drug discovery and development
were clearly visible and quantifiable in the electropherogram in
Figure 4 (upper panel). The inset shows the full-scale image ofthe separation. The reproducibility of this sample preparation

Measurement

protocol is depicted in Table 1. Figure 4 (lower panel) delineates
the evaluation of the workflow for CESI-MS analysis featuring
the total ion electropherogram of the PNGase F released and
APTS labeled N-linked human serum oligosaccharides. All glycan

CE-LIF

structures identified by CE-LIF and CESI-MS methods are shownin Table 2 with the corresponding structural information and theirintegrated peak area values.
CESI-MS%RSD
Intra-dayInter-day

Hardware performance(peak area)

0.92

1.35

hIgG1 sample preparation(peak area)

3.40

5.42

Human serum sample
preparation (peak area)

7.83

9.63

Peak area (total)

6.11

8.53

Migration time

1.42

2.31

Table 1. Reproducibility of the sample preparation scale-up andanalysis workflow.

Conclusions
Based on the significant sample preparation improvementsof the individual optimized steps described in this technicalnote, a novel workflow was established to analyze the N-linked
carbohydrates of complex biological samples readily applicablefor bioprocessing specimens. The sample preparation scaleup protocol was applied for N-linked glycosylation analysis
of human serum samples without any precipitation-related
problems up to 1.5 mg protein content by using the combinationof amine-functionalized magnetic bead-based protein capture
and temperature gradient denaturation with our earlier reported
evaporative labeling protocol. In addition, this approachefficiently removed the free sugar content of complex biologicalsamples (e.g., serum glucose) that would otherwise inhibit theendoglycosidase digestion reaction during N-glycan release.
Deep N-glycomic analysis of human serum samples wasdemonstrated using CE-LIF and CESI-MS methods, and
28 individual structures were identified.

Figure 4. Capillary electrophoresis separation of PNGase F released
and APTS labeled human serum N-glycans by CE-LIF (upper panel)
and CESI-MS (lower panel). Separation conditions: CE-LIF, same as in
Figure 2; injection, 1 kV/1 s; CESI-MS, OptiMS cartridge (91 cm total
length, 30 μm ID/150 μm OD); background electrolyte, 10 mM ammonium
acetate, 0.1% acetic acid (pH 4.5) and 20% isopropanol; applied voltage:
E=220 V/cm in reversed polarity mode at 20 °C with additional 2 psi
forward pressure at both the separation and the conductive lines; injection,
1 psi/5 s water pre-injection followed by 10 kV/20 s sample injection.

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Drug discovery and development

No.

Abbreviated structuralinterpretation

Glycan composition

Neutral mass

Doubly charged
APTS labeled mass

1

A2G2S2

H5N4A22222.781330.87

2

A2BG2S2

H3N51519.57

979.26

3

FA2G2S2

H5N4A2F1

2368.841403.89

4

FA2BG2S2

H5N5A2F1

2571.921505.43

5

A2G1S1

H4N4A1

1769.631104.29

6

A3G3S2

H6N5A22587.92

1513.43

7

FA2G1S1

H4N4A1F1

1915.69

1177.32

8

A2G2S1

H5N4A1

1931.69

1185.32

9A2BG2S1

H5N5A1

2134.77

1286.8610

FA2G2S1

H5N4A1F1

2077.75

1258.35

11

A2

H3N4

1316.49877.72

12

M5

H5N2

1234.43836.6913

FA2BG2S1

H5N5A1F1

2280.821359.89

14

A2B

H3N5

1519.57

979.2615FA2

H3N4F11462.54950.75

16

M6

H6N2

1396.49917.72

17

A2G1

H4N4

1478.54958.7418

FA2B

H3N5F1

1665.621052.29

19

A2BG1

H4N5

1681.621060.28

Glycan structurep5

Drug discovery and development

20

FA2G1

H4N4F1

1624.60

1031.7721

FA2BG1

H4N5F1

1827.68

1133.3122

A4G4S1

H7N6A12661.95

1550.45

23

A2G2

H5N4

1640.591039.77

24

A2BG2

H5N5

1843.67

1141.3125

FA2G2

H5N4F1

1786.651112.8026

FA2BG2

H5N5F1

1989.73

1214.34

27

A3G3

H6N5

2005.721222.34

28

A4G4

H7N6

2370.861404.90

Table 2. Identified N-glycan structures by CE-LIF and CESI-MS.5

Acknowledgements
The authors gratefully acknowledge the support of the National
Research, Development and Innovation Office (NKFIH)(K 116263) grants of the Hungarian government and the
EFOP-3.6.3-VEKOP-16-2017-00009 program co-financed by
the EU and the European Social Fund.

References
1. Anderson, N.L.; Anderson, N.G. The human plasma proteome:history, character, and diagnostic prospects. Mol Cell
Proteomics. 2002, 1(11), 845–67.

2. Varki, A. et al. Essentials of Glycobiology, 2nd ed.; Cold Spring
Harbor Laboratory Press (Plainview, NY), 2009.
3. Reider, B.; Szigeti, M.; Guttman, A. Evaporative fluorophorelabeling of carbohydrates via reductive amination. Talanta.
2018, 185, 365–9.
4. Szigeti, M.; Guttman, A. Sample preparation scale-up fordeep N-glycomic analysis of human serum by capillaryelectrophoresis and CE-ESI-MS. Mol Cell Proteomics.
2019, 18(12), 2524–31.
5. Harvey, D.J. et al. Symbol nomenclature for representing
glycan structures: Extension to cover different carbohydrate
types. Proteomics, 2011, 11(22), 4291–5.

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