Development of an Instant Glycan
Labeling Dye for High Throughput
Analysis by Mass Spectrometry
Michael Kimzey, Zoltan Szabo, Vaishali Sharma,
Alexander Gyenes, Samnang Tep, Adele Taylor,
Aled Jones, Justin Hyche, Ted Haxo, Sergey Vlasenko
www.prozyme.com
Development of an Instant Glycan Labeling Dye for High Throughput Analysis by Mass Spectrometry
INTRODUCTION
RESULTS
Glycosylation of biotherapeutic proteins is frequently a critical
quality attribute; therefore the characterization of glycans on
biotherapeutics is an important activity in the development
process. A common approach is to release and label N-glycans
with a tag to allow for fluorescence detection; a process that
often requires numerous hours to complete. Unfortunately,
many of the most commonly used fluorescent tags have poor
MS sensitivity.
• We present a novel instant glycan labeling reagent,
InstantPC™ (IPC) that provides markedly increased MS and
FLR sensitivity
• The workflow utilizes a rapid in-solution digestion, instant
labeling, and cleanup of excess IPC label
• IPC is shown to lend itself to both rapid (5-minute) and
high-resolution (60-minute) HILIC methods for N-glycan
separation, allowing flexibility for screening applications
and in-depth characterization
Structural features of IPC.
The IPC structure (Figure
1) is an activated form of
Procaine [2,3,4], which labels
glycosylamines released by
PNGase F digestion. IPC
attachment forms a stable urea
linkage with the N-glycan.
O
O
O
N O
O
O
N
N
H
Figure 1: Structure of InstantPC.
A novel instant glycan label for
MS [2,3,4].
Sample preparation. Glycoprotein preparation with IPC
uses an N-Glycanase digestion time of 5 minutes. The short
digestion time was enabled by ProZyme’s new proprietary
methods, which use denaturants that are both enzyme- and
MS- friendly [1]. The denaturant is convenient and rugged, as
the solution is stable at room temperature and does not require
special handling procedures. To illustrate the cleanup, a mixture
of human IgG and bovine fetuin was labeled with IPC. Half of
the samples went through the cleanup and the other half did
not. Figures 2A and 2B show unbiased and near-complete
recovery of this complex mixture of IPC-labeled glycans.
METHODS
Materials
Enbrel® lot # 1036862, Herceptin® lot # B1629B11
A
N-Glycanase digestion, IPC labeling and cleanup
A developmental protocol was used to release N-glycans with
a 5-minute in-solution digest. In ProZyme’s proprietary method
[1], a solution of 20 µl of glycoprotein at 2 mg/ml (40 µg total)
was treated with a shelf-stable master mix consisting of buffer,
reductant, and denaturant. The sample was briefly heated,
prior to incubation with N-Glycanase for 5 minutes at 50 °C.
Released glycosylamines were labeled with 5 µl of IPC, and
the labeled N-glycans were cleaned up and eluted in aqueous
buffer using a modified protocol for GlykoPrep® CU cartridges.
Before Cleanup
5-Minute screening UHPLC method
Agilent AdvanceBio Glycan Mapping column, 2.1 x 100 mm
2.7 µm, flow rate 1.4 ml/min, 23–40% 100 mM Ammonium
Formate pH 4.4 in 4 minutes, column temperature 35 °C,
excitation 285 nm, emission 345 nm.
After Cleanup
0
2
4
6
B
MS/MS conditions
Collision energy ramp of 40–60 V for +1; 15–30 V for +2;
15‑25 V for +3; 1.0 second scan time, m/z range 50–2000 Da.
90
1E+8
85
80
8E+7
75
6E+7
70
65
4E+7
60
2E+7
% Sialylated (points)
MS conditions
Waters Xevo G2-S QTof, + mode, capillary voltage 2.8 kV,
cone voltage 30 V, source temperature 120 °C, desolvation
temperature 350 °C, scan time 0.8 second, m/z range
300–2000 Da.
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Minutes
Total N-glycan RFU signal (bars)
60-Minute high-resolution UHPLC method
Agilent AdvanceBio Glycan Mapping column, 2.1 x 150 mm
2.7 µm, flow rate 0.4 ml/min, 20–46% 50 mM Ammonium
Formate pH 4.4 in 43.5 minutes, column temperature 45 °C,
excitation 285 nm, emission 345 nm.
55
0E+0
Before
Cleanup
After
Cleanup
50
Figure 2: Cleanup of IPC-labeled N-glycans. (A) HILIC profiles
of N-glycans from human IgG (75%) and bovine fetuin (25%),
± cleanup. Neutral peaks 10–18 minutes, sialylated peaks 18–30
minutes. (B) Triplicate samples before and after cleanup of free
IPC. Total glycan fluorescence in blue bars, % sialylated peaks on
secondary axis.
2
UHPLC performance. One of the features of IPC compared to
Procainamide or 2-AB is that IPC is less polar, so IPC-labeled
glycans elute earlier on HILIC. This results in shorter retention
times, so the gradient must be adjusted to start with a higher
percentage of acetonitrile. AdvanceBio Glycan Mapping
columns (2.7 µm) are particularly useful for IPC-labeled
N-glycan analysis, as they provide a key separation of Man5
from G1[6] which may be important for screening mAbs during
cell-culture optimization (Figure 3). If greater resolution is
required, a 60-minute gradient may be used (Figure 4).
Man9
Man8
G2F
Man7
G0F
A1
A2
G2F
3.7
Figure 3: 5-minute HILIC separation of IPC-labeled N-glycans.
(A) mixture of high mannose and asialo biantennary N-glycans, (B)
Enbrel, and (C) Herceptin.
1.5E+5
1.0E+5
5.0E+4
C
tP
uo
an
Fl
st
pi
In
Pr
Ra
Extracted MS peak area of G0F
Man9
Man8
Man9
Man8+A2F
A2
1.5E+5
1.0E+5
5.0E+4
B
id
m
Pr
oc
ai
na
uo
Fl
Ra
2A
e
S
r- M
C
tP
an
A2
A1F[6]
A2F
In
st
A1[3]
A1[6]
G1FS1[3]
G2F
G2
G1F[3]
2.0E+5
0.0E+0
A1F[3]
Man7/1
Man7/2
A1F[6]
A1F[3]
G1FS1[3]
G2F
G1S1[6]
Man6
G1S1[3]
G2 + G1FS1[6]
G1F[6]
G1F[3]
G1[6]
G1[3]
G1[6]
G1[3]
2.5E+5
pi
G2F
Man7/1
Man7/2
G2
G1F[3]
Man6
G1F[6]
G1[3]
G1[6]
G0F
Man5
Man5
Man5
G0
G1F[6]
C
Enbrel N-glycans
G0F
G0
B
High mannose and
biantennary complex
N-glycans with
2,3-linked sialic acid
MS
3.0E+5
G0F
G0
A
N-glycan mixture,
asialo and high
mannose
B
0.0E+0
2A
3.5
e
3.3
id
3.1
na
2.9
ai
2.5 2.7
Minutes
2.0E+5
oc
G1F[6]
G1F[3]
2.3
2.5E+5
S
2.1
G1F-N
Man5
1.9
FLR Peak area of G0F
1.7
G0F-N
G0
G0-N
1.5
Fluorescence
3.0E+5
G0F
m
G1F[3]
A2F
Man6
G2
G1[6]
G1[3]
Man5
G1F[6]
G2F + G1FS1
A1F
G0
C
Herceptin
1.3
Man6
G2
Man5
G1[6]
G1[3]
G1F[6]
G1F[3]
G0F
G0
B
Enbrel
Comparison to other labels. IPC has the highest LC-fluorescence
of all glycan labels tested. The next best label for fluorescence
was Procainamide, which was prepared by reductive amination
(Figure 5). The MS response of IPC was comparable to the
glycosylamine reactive label RapiFluor-MS, yet RapiFluor-MS is
not suitable for screening using this method, as Man5 does not
separate from G1 (Figure 7 - see next page). Software programs
such as UNIFI are not recommended for quantification of
coeluting species such as Man5/G1[6] because it will assign the
relative peak area to the most intense m/z only.
r- M
A
Mixture of high mannose
and asialo biantennary
N-glycans
MS performance. IPC contains a tertiary amine which generates
high MS signal in positive mode. IPC will add a monoisotopic
mass of 261.14773 Da to a reducing end. Most commonly used
labels for glycan analysis ionize poorly, so FLR is typically the only
choice for analysis of low abundance glycans. IPC-labeled glycans,
however, have such an improved ionization efficiency that the
MS signal is comparable to or better than FLR sensitivity. For the
N-glycans on Enbrel, most IPC-labeled biantennary glycan ions
are [M+2H]2+ using the conditions provided.
Figure 5: FLR and MS Response Comparison. Response of
RapiFluor-MS, Procainamide and 2-AB labeled G0F.
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Minutes
Figure 4: 60-minute HILIC separation of IPC-labeled N-glycans.
(A) high mannose and asialo biantennary structures, (B) high mannose
and biantennary complex structures with 2,3-linked sialic acid and
(C) N-glycans from Enbrel.
3
Development of an Instant Glycan Labeling Dye for High Throughput Analysis by Mass Spectrometry
IPC
IPC
IPC
100 x6
IPC
IPC
IPC
–H 2 0
IPC
IPC
IPC
IPC
IPC
IPC
IPC
IPC
IPC
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050 1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700 1750
1800
1850
1900
1950
2000
2050
2100
2150
IPC
200
IPC
150
IPC
100
IPC
IPC
IPC
IPC
IPC
–H 2 0
50
IPC
–H 2 0
0
2200 2250
2300
2350
2400
2450
2500
2550
2600
2650
m/z
Figure 6: CID MS/MS of IPC-labeled A2F
MS/MS. IPC is suitable for Collision Induced Dissociation MS/MS.
As with other positively charged tags such as Procainamide, the
CID profile contains mostly glycosidic cleavages with some crossring fragmentation. A2F (aka G2FS2 or FA2G2S2) from Enbrel
is a disialylated fucosylated biantennary glycan with α2,3-linked
NANA (Figure 6).
both IPC and RapiFluor-MS, however, share the labeling artifact that
is apparent for afucosylated reducing end GlcNAc (Figures 8 and
9). This is a feature of GlcNAc glycosylamine labeling and efforts to
understand this artifact are underway.
IPC
Man5
RapiFluor-MS
*
G1[6]
G1[3]
*
*
A
B
A
B
*
C
C
IPC
1.1
12 13 14 15 16 17 18 19 20 21 22 23
Minutes
1.5
1.9
2.3
2.7
3.1
Minutes
3.5
3.9
4.3
Figure 8: Glycosylamine labeling artifact. Extracted ion
chromatograms from (A) G2 and (B) Man5 N-glycans show the
labeling artifact (*) peak for Enbrel. FLR trace is shown in (C). Left is
IPC, right is RapiFluor-MS. The artifact is ~1.5% of main peak and
is consistent across both glycosylamine reactive labels. This artifact
is typically observed only in high resolution methods and coelutes
with parent peak with screening and medium resolution gradients.
Man5/G1[6]
G1[3]
Figure 9: GlcNAc Glycosylamine.
The early artifact peak results from an
isomer, likely at C1 (*) or an alternative
chair conformation of the reducing end
glycoslamine ring. C2 epimers (GlcNAc
to ManNAc) may be ruled out as they
result in later LC retention times.
RapiFluor-MS
1
3
5
7
9
11
13 15
Minutes
IPC - 5-minute method
2
6
10
14
18
22
12 13 14 15 16 17 18 19 20 21 22
Minutes
17
19
21
23
25
OH
O
Glycan
HO
*
NH2
NH
O
RapiFluor-MS
55 minute method
26 30 34
Minutes
38
42
46
50
CONCLUSIONS
54
1 IPC has the highest fluorescence response of any glycan
label tested
2 IPC shows high MS response, superior to Procainamide,
and comparable to RapiFluor-MS
3 IPC rapid LC methods separate Man5 from G1 for mAb
screening
4 New 5-minute deglycosylation method using MS
friendly denaturant enables both screening and in-depth
characterization on N-glycans using a single workflow
Figure 7: Run Time and Resolution Comparison. Resolution
of Man5, G1[6], and G1[3] IPC-labeled glycans vs. RapiFluor-MS
labeled glycans. IPC-labeled glycans using AdvancedBio Glycan
Mapping column vs. RapiFluor-MS labeled glycans using Glycan
BEH column.
REFERENCES
1. Patent Pending
2. US Patent 8124792B2
3. US Patent 8445292B2
4. Patent Pending
ProZyme, GlykoPrep, N-Glycanase and InstantPC are registered trademarks of ProZyme,
Inc. in the United States and other countries. All other trademarks are the property of their
respective owners.
4