Comprehensive quali-quantitative profiling of neutral and sialylated: O -glycome by mass spectrometry based on oligosaccharide metabolic engineering and isotopic labeling

Article abstract of DOI:10.1039/c9ra01114e

Mass spectrometry (MS) analysis combined with stable isotopic labeling is of great importance for quantitatively profiling abnormal sialylated O-glycans associated with disease development, but technically hindered by the poor releasing efficiency of O-gl

Full text of DOI:10.1039/c9ra01114e

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Comprehensive quali-quantitative profiling of
neutral and sialylated O-glycome by mass
spectrometry based on oligosaccharide metabolic
engineering and isotopic labeling†
Cite this: RSC Adv., 2019, 9, 15694
a
ab
ab
ab
ab
ab
Lijing Nan, Jiao Li, Wanjun Jin, Ming Wei, Mengjun Tang, Chengjian Wang,
ab
ab
a
ab
Guiping Gong, Linjuan Huang, Ying Zhang* and Zhongfu Wang
*
Mass spectrometry (MS) analysis combined with stable isotopic labeling is of great importance for
quantitatively profiling abnormal sialylated O-glycans associated with disease development, but
technically hindered by the poor releasing efficiency of O-glycans from glycoprotein or the labile nature
of sialic acid residues at glycans. Herein, we developed an isotopic precursor based metabolic
amplification and labeling (IPMAL) technique for relative quantitative profiling of the repertoire O-glycans
between normal and tumor cells by ESI-MS. Two groups of cells were incubated with peracetylated
d0
benzyl-a-N-acetylgalactosamine (Ac
3
GalNAc-a-Bn ) or a heavy labeled peracetylated benzyl-a-N-
d5
3
acetylgalactosamine (Ac GalNAc-a-Bn ) precursor respectively to amplify the repertoire of O-glycans
d0/d5
as Bn
-O-glycans which could achieve the quantitative O-glycome analysis by ESI-MS after
derivatization. The established method demonstrates desirable feasibility, accuracy (relative error (RE) #
2
4
.20%), reproducibility (coefficient of variation (CV) # 7.61%, n ¼ 3) and good quantitation linearity (R
>
0
.99, n ¼ 3) for five Bn-O-glycans with 2 orders of magnitude. Finally, the method has been successfully
Received 12th February 2019
Accepted 11th May 2019
applied to quantitative analysis of the repertoire O-glycome changes between normal human liver cell
line L02 and human hepatoma cell line SMMC-7721. Moreover, the a-2,3/2,6 sialic acid isomers of Bn-
O-glycans from these two cells have been further quantitatively distinguished when involved a sialic acid
specific derivatization procedure.
DOI: 10.1039/c9ra01114e
rsc.li/rsc-advances
determination as well as sialic acid linkage specic identica-
tion of O-glycans expressed in normal and tumor cells will be
Introduction
Mucin-type O-glycans at glycoproteins are covalently attached to benecial for functional elucidation and diagnostic or prog-
Ser/Thr residues of the core protein through the GalNAc residue nostic purposes during disease development procedures as
and play important roles in a variety of biological processes required in pathology or glycomics.
such as inter-cellular and intra-cellular interactions, commu-
nication, disease development and signal transductions.
Mass spectrometry with high sensitivity and resolution, has
played an important role in the structural analysis of glyco-
1
–3
Terminal a-2,3 and/or a-2,6 sialic acids (N-acetylneuraminic conjugates and quantitative analysis of glycans changes
acid or N-glycolylneuraminic acid) at O-glycans have shown between two samples using “light” and “heavy” stable isotopic
12–14
signicant biological importance in receptor recognition labels.
One common method employing NaOH solution to
processes such as inammation, bacterial and virus infec- release and instantaneously reduce the aldehyde ends at O-
4
–7
tions. Moreover, aberrant O-glycans at glycoprotein with or glycans with “light” and “heavy” isotopic reagents, NaBH
without sialic acids in both structures and their expression NaBD , respectively, has been widely used for MS quantication
levels are associated with various human diseases such as proling of O-glycome changes between two samples. Besides,
4
and
4
15
8–11
tumors or cancer.
Therefore, the quali-quantitative the O-glycans derived from two samples could be permethylated
1
2
13
with CH I/ CH I or CD I/CH I isotopic labeling strategy to
3
3
3
3
achieve the quantitation determination of overall O-glycome by
a
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of
Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences,
16,17
18
MS.
Wang et al. have developed an one-pot O-glycans
releasing and d0/d5-PMP isotope labeling assay for qualitative
College of Food Science and Technology, Northwest University, Xi'an 710069, P. R. and quantitative proling of O-glycans from glycoproteins by
Northwest University, Xi'an 710069, P. R. China. E-mail: wangzhf@nwu.edu.cn
b
China
MS. Recently, Kudelka had reported an isotopic labeling with
cellular O-glycome reporter/amplication (ICORA) for
†
Electronic supplementary information (ESI) available. See DOI:
1
0.1039/c9ra01114e
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19
comparative O-glycomics of cultured cells by MS. Despite all
progress in quantication determination of O-glycans in gly-
comics studies, further investigation is needed to be conducted
to improve/resolve these corresponding defects, such as
misunderstanding of natural peaks of glycans with the isotope-
labeled one due to the small molecular weight difference (1 Da),
lack of universal hydrolytic enzyme used for effectively releasing
of all O-glycans from glycoproteins, large amounts of glyco-
proteins used which might be not practicable for biological
sample analysis and difficulty in quali-quantitative identica-
tion of a-2,3/2,6 sialic acid terminal ends of O-glycan isomers by
20–22
MS.
Although mass spectrometry has become an important tool to
determine glycan structures, poor ionization of native glycans or
sialic acids signal loss at glycans were easily happen when
23,24
without a derivatization procedure.
Moreover, MS and/or MS/
MS glycomics proling might face challenge in quali-quantitative
determination of a-2,3/2,6-linked sialic acids at the terminal ends
25
of glycan isomers. Due to the pathological importance of the O-
26
glycan terminal sialic acids, it is of great signicance to estab-
lish a high-throughput and effective method for MS-based
quantication of overall neutral and sialylated O-glycans with
linkage specicity between normal and tumor cells.
In our previous work, a sialic acid specic derivatization
combined with Girard's reagent T (GT) on-target labeling has
been exploited for MS-based linkage specic determination of
27
sialic acids at kinds of glycans. Importantly, it has been re-
ported recently the dimethylamidation is compatible with
traditional permethylation to achieve the linkage-specic Scheme 1 Isotopic precursor based metabolic amplification and
28
labeling (IPMAL) strategy for quantitatively profiling the repertoire of
O-glycans between normal and tumor cells. Structure formulas:
yellow square, N-acetylgalactosamine (GalNAc); blue square, N-ace-
tylglucosamine (GalNAc); yellow circle, galactose (Gal); red triangle,
fucose (Fuc); purple diamond, sialic acid (N-acetylneuraminic acid,
identication of sialic acids at N-glycan ends by MS. These
research ndings inspire us to utilize the strengths of chemical
derivatization and oligosaccharide metabolic engineering and
isotopic labeling strategy, to develop an isotopic precursor
based metabolic amplication and labeling (IPMAL) assay for Neu5Ac; N-glycolylneuraminic acid, Neu5Gc).
quali-quantitatively proling of sialic acids linkage specicity of
29
O-glycans between normal and tumor cells by MS.
alcohol,
dimethylamine
hydrochloride,
1-ethyl-3-(3-
Within our IPMAL strategy, normal and tumor cells were (dimethylamino)propyl)-carbodiimide (EDC) hydrochloride and
d0
3 3
respectively incubated with Ac GalNAc-a-Bn and Ac
GalNAc-a- 1-hydroxybenzotriazole (HOBt) monohydrate were purchased from
d5
Bn (5 Da mass difference) substrates, which could enter cells and Aladdin Inc. (Shanghai, China). Dimethyl sulfoxide, benzyl-
be used by enzymes in Golgi to amplify the repertoire of O-glycome 2,3,4,5,6-d5 alcohol and 4-ethynylbenzyl alcohol were purchased
19,29
to be Bn-O-glycans.
Comprehensive quali-quantitative proling from Sigma-Aldrich (St. Louis, MO, USA). High-glucose Dulbecco's
of Bn-O-glycans from two cells aer 1 : 1 mixing could be achieved modied Eagle's medium (DMEM) was purchased from HyClone
by MS, as illustrated in Scheme 1. We have evaluated the method (Logan, UT, USA). Amicon Ultra 4 mL centrifugal lters (10 kDa)
0
in terms of feasibility, reproducibility, accuracy and linear prop- were purchased from Millipore (Bedford, MA, USA). 3 -Sialyllactose
0
0
0
erties. The established assay has been applied to quali- (3 -SL, a2,3-linked sialic acid) and 6 -sialyllactose (6 -SL, a2,6-
quantitatively prole and determine the O-glycans between linked sialic acid) were purchased from Carbosynth (Compton,
normal human liver cell line L02 and human hepatoma cell line U.K.). Sep-Pak C18 (3 cc) solid phase extraction (SPE) cartridges
30,31
SMMC-7721 aer permethylation.
Furthermore, the whole O- were obtained from Water Corporation (Milford, MA, USA).
glycan isomers with/without a-2,3/2,6 sialic acid ends have been Human hepatoma cells SMMC-7721 and human normal liver cells
quali-quantitatively distinguished when a sialic acid specic L02 cell lines were purchased from Shanghai cell bank, Chinese
28
1
13
derivatization procedure was involved.
Academy of Sciences. H and C NMR spectra were recorded with
Bruker AC-600 spectrometers using CDCl as solvent. Mass spectra
3
were measured under a Thermo Fisher Scientic LTQ Orbitrap XL
spectrometer. The chemical reaction was monitored by thin layer
chromatography (silica gel, 25 mm layer thickness) and detected by
tri- a UV lamp (254 nm) combined with sulfuric acid-lichen phenol
Materials and methods
Chemicals and materials
N-Acetyl-D-galactosamine,
1,2-dichloroethane,
copper
uoromethanesulfonate (Cu(OTf)
), benzyl alcohol, 4-nitrobenzyl staining. All of these reagents were of analytical grade.
2
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d0
d5
1
Synthesis of O-glycan precursors Ac
3
GalNAc-a-Bn and
Ac
CDCl
3
1
3
GalNAc-a-Bn , purity 97.8% (HPLC); H NMR (600 MHz,
), d 7.26 (s, 6H), 5.56 (d, J ¼ 9.6 Hz, 1H), 5.37 (t, J ¼ 9.8 Hz,
d5
Ac
3
GalNAc-a-Bn
H), 5.18 (dd, J ¼ 11.3, 3.1 Hz, 1H), 4.98 (d, J ¼ 3.5 Hz, 1H), 4.72
Synthesis of O-glycan precursors were carried out according to
3
2–34
(d, J ¼ 11.7 Hz, 1H), 4.64–4.53 (m, 1H), 4.50 (d, J ¼ 11.7 Hz, 1H),
a reported procedure with minor modication.
of glycosamine pentaacetates (200 mg, 0.514 mmol) in dry 1,2-
dichloroethane (10 mL), anhydrous Cu(OTf) (0.15 equiv.) and
d0-benzyl alcohol (4 equiv.) or heavy labeled benzyl alcohol
benzyl-2,3,4,5,6-d5 alcohol, 4 equiv.) were added. The reaction
To a solution
4
2
.22 (t, J ¼ 6.4 Hz, 2H), 4.10 (m, 2H), 2.16 (s, 3H), 2.06 (d, J ¼
13
3
.2 Hz, 3H), 1.95 (s, 2H), 1.90 (s, 3H). C NMR (151 MHz, CDCl )
2
d 173.56, 173.04, 172.97, 172.59, 102.31, 99.65, 79.87, 79.66,
9.45, 77.12, 72.72, 71.09, 70.02, 69.60, 64.55, 63.03, 50.39,
5.92, 23.69, 23.40, 23.33. ESI-MS: calcd for C21 [M +
7
2
(
H32NO
9
mixture was stirred vigorously under an N atmosphere reux at
30 C for 6 h. When the reaction was completed, the saturated
2
+
ꢀ
Na] m/z 465.08, found m/z 465.17.
1
NaHCO
3
aqueous solution was added to the reaction mixture to
Cell culture
stop the reaction. The organic layer was totally layered aer
stirring. The collected organic layer was concentrated under the Cell culture was carried out according to the reported proce-
35
reduced pressure aer washing with water twice. The concen- dure. SMMC-7721 and L02 cells were cultured with 10 mL
trated organic residues were concentrated and loaded onto DMEM medium containing 10% (by volume) fetal bovine serum
ꢁ
1
silica gel column separation using petroleum ether–EtOAc (1 : 1 and 100 U mL penicillin–streptomycin mix. Cells were kept in
ꢀ
or other ratios based on TLC separation result) as mobile phase an incubator at 37 C, 5% CO and 95% (volume fraction) air-
2
d0
to acquire the desired product Ac GalNAc-a-Bn (R ¼ 0.75, saturated humidity. When the cell density was increased to
3
f
d5
7
1
7
61.77 mg, 72%) or Ac
3
GalNAc-a-Bn (R
f
¼ 0.75, 161.35 mg, 80% (cells 2 ꢂ 10 ), the medium with 10% fetal bovine serum
1%), which were further conrmed by MS and NMR data, as was replaced with medium containing 8% (volume fraction)
presented in Fig. 1B and S1A–D (ESI† le).
fetal bovine serum. Then Ac GalNAc-a-Bn (100 mM) was added
3
d0
1
Ac
CDCl
3
GalNAc-a-Bn , purity 98.6% (HPLC); H NMR (600 MHz, to medium and kept for further three days.
), d 7.37 (d, J ¼ 7.5 Hz, 2H), 7.31 (d, J ¼ 6.8 Hz, 1H), 7.26 (s,
3
1
1
1
4
2
1
7
2
H), 5.58 (d, J ¼ 9.7 Hz, 1H), 5.38 (d, J ¼ 2.5 Hz, 1H), 5.17 (dd, J ¼
Acquisition and purication of Bn-O-glycans
1.3, 3.2 Hz, 1H), 4.97 (d, J ¼ 3.6 Hz, 1H), 4.72 (d, J ¼ 11.7 Hz,
H), 4.61–4.57 (m, 1H), 4.49 (d, J ¼ 11.7 Hz, 1H), 4.21 (s, 1H),
.09 (dd, J ¼ 18.6, 6.6 Hz, 2H), 2.16 (s, 2H), 2.05 (s, 2H), 1.98 (s,
Acquisition and purication of Bn-O-glycans were carried out
29
according to reported procedure with minor modication.
1
3
Aer three days of culture, the culture medium was collected
and ltered through a 10 kDa ultraltration membrane (2500g,
3
H), 1.90 (s, 3H). C NMR (151 MHz, CDCl ) d 173.59, 173.09,
73.02, 172.63, 139.20, 131.35, 131.04, 130.89, 99.63, 79.90,
9.68, 79.47, 72.77, 71.06, 70.00, 69.58, 64.56, 50.34, 25.95,
3.43, 23.40. ESI-MS: calcd for C21
30 min). The ltered solution was puried through a C18
+
cartridge (200 mg/4 mL) according to the following procedure.
The column was equilibrated with 4 mL of acetonitrile, 8 mL of
H27NO
9
, [M + Na] m/z 460.35,
found m/z 460.08.
50% (v/v) acetonitrile and 10 mL of 0.1% (v/v) triuoracetic acid
(TFA). Then the ltered media was loaded onto the column and
washed with 2 mL of 0.1% (v/v) triuoracetic acid (TFA) four
times. Bn-O-glycans were then eluted with 3 mL of 50% (v/v)
acetonitrile containing 0.1% TFA. The puried sample was
collected and lyophilized to dry.
General permethylation of Bn-O-glycans
General permethylation of O-glycan samples was carried out
according to the reported procedures with minor modica-
3
0,31
tion.
The dried sample was dissolved in 300 mL DMSO–NaOH
I mixture. Aer shaking 30 min in
suspension and 75 mL CH
3
dark, 500 mL water was added into sample to quench the reaction.
Then 500 mL dichloromethane was added. The supernatant was
then partitioned between water and dichloromethane. The
organic layer was washed with 500 mL water twice, dried and re-
dissolved in 200 mL 50% methanol for further MS analysis.
The whole sialic acid specic derivatization procedure for Bn-
O-glycans
The whole derivatization procedure has two steps, including
linkage specic derivatization of sialic acid and general per-
methylation. Linkage specic derivatization of sialic acid was
conducted according to the previous references with minor
3
Fig. 1 (A) Synthetic scheme for Ac GalNAc-a-Bn precursor and (B)
d0
3 3
ESI-MS spectrum of the synthesized Ac GalNAc-a-Bn and Ac -
d5
GalNAc-a-Bn
.
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ꢀ
25 mL of 500 mM 1-hydroxybenzotriazole temperature of 300 C, positive ion mode scan, scan range is m/z
modication.
(HOBt, dissolved in DMSO), 25 mL of 250 mM 1-ethyl-3-(3- 200–2000. MS/MS analysis was carried out using helium (He) as
dimethylaminopropyl) carbodiimide hydrochloride (EDC, dis- the collision gas, a normalized collision energy degree of 35–
solved in DMSO) and 25 mL of 250 mM dimethylamine dissolved 45% and an isotope width of m/z 2.00. The ESI-MS and MS/MS
in DMSO, were added to the dried glycan sample (5 pmol to 10 data were acquired with LTQ Tune soware Xcalibur (Thermo).
ꢀ
nmol) and incubated at 60 C for 1 h. The excess dimethyamine Other parameters were acquiescent.
ꢀ
was evaporated in a Speed Vac at 60 C for 20 min. Then the
sample was treated with 200 mL of NaOH–DMSO slurry and 100
mL of methyl iodide for 30 min with agitation at room temper-
Results and discussions
ature. The reaction was terminated by adding 800 mL of water. Kudelka recently have developed an isotopic labeling with
Subsequently, 200 mL of dichloromethane was added. The cellular O-glycome reporter/amplication (ICORA) strategy,
H7
mixture was stirred and centrifuged to facilitate partitioning. which employed two O-glycan precursors, Ac GalNAc-Bn and
3
D7 19
The top aqueous layer was then removed, while the dichloro- Ac GalNAc-Bn
. The assay provides a useful tool for exploring
3
methane layer was washed with 1 mL water three times addi- the dynamic changes in O-glycan between two cells. However,
tionally. Then the organic layer was dried and submitted to the the incapability of determination a2,3/2,6-sialylated O-glycan
general permethylation procedure as described above.
isomers with linkage specicity by MS and/or MS/MS might be
limit its application at some extent. Moreover, dimethylami-
dation has been used to distinguish a-2,3/2,6-linked sialic acids
at N-glycans, which is compatible with common per-
methylation. Herein, an innovative strategy for the rst MS-
based quali-quantitative proling and linkage-specic identi-
cation of O-glycans with or without sialic acids ends through
isotopic precursor based metabolic amplication and labeling
Feasibility evaluation of the assay
Two groups of human hepatoma cells SMMC-7721 cells were
28
d0
3 3
parallelly cultured with 100 mM Ac GalNAc-a-Bn and Ac -
d5
GalNAc-a-Bn , respectively, for 3 days. Both cell media was
collected and mixed at a ratio of 1 : 1. The mixed culture
medium was then submitted to purication, permethylation
and MS-based quantication analysis.
(
IPMAL) assay could be achieved, when involved the sialic acid
28
specic derivatization.
Evaluation the linearity of the assay
Principle of the assay
Two groups of SMMC-7721 cells were in parallel cultured with
A schematic procedure for quantitative proling of the reper-
toire of O-glycome through isotopic precursor based metabolic
amplication and labeling (IPMAL) assay, is depicted in Scheme
d0
d5
1
00 mM Ac GalNAc-a-Bn and Ac GalNAc-a-Bn , respectively,
3
3
for 3 days. The media from two conditions was mixed in
a gradient ratios (10 : 1, 7.5 : 1, 5 : 1, 2.5 : 1, 1 : 1, 1 : 2.5, 1 : 5,
1. We designed a novel strategy for rapid synthesis of the
1
: 7.5 and 1 : 10, n ¼ 3). Linearity evaluation of the assay was
normal and/or heavy labeled precursors, as depicted in Fig. 1A.
One step O-glycosylation of glycosamine pentaacetates with
a normal or heavy labeled benzyl alcohol could afford the nal
plotted using relative intensities ratios of major O-glycan pair
peaks observed in MS spectra as the y-axis versus the adding
linear gradient ratios (10 : 1, 7.5 : 1, 5 : 1, 2.5 : 1, 1 : 1, 1 : 2.5,
d0
d5
3 3
products Ac GalNAc-a-Bn or Ac GalNAc-a-Bn in good yields
1
: 5, 1 : 7.5 and 1 : 10) of O-glycans mixture as the x-axis. The
1
13
(>70%), as conrmed by NMR (Fig. S1A–D, H and C NMR,
calibration graphs for the assay were calculated by the equation:
y ¼ mx + c.
ESI† le) and ESI-MS (Fig. 1B). Both neutral and sialylated O-
glycans could be amplied through oligosaccharide metabolic
d0
engineering in the presence of precursors Ac GalNAc-a-Bn or
3
Statistical analysis
d5
Ac GalNAc-a-Bn (5 Da mass difference) within two cells. Then
3
Results of all experiments performed for the relative intensity quali-quantitative proling of O-glycans as well as sialic acid
ratios of peak pairs in MS-based relative quantication in trip- linkage specic characterization of O-glycans could be achieved
29
licates were indicated by mean ꢃ SD. Data was analyzed by one- by MS and MS/MS when involved a permethylation or the
28
way analysis of variance (ANOVA) using SPSS 16.0. Statistical sialic acid specic derivatization procedure, as illustrated in
differences were determined using a two-tailed Student's t-test, Fig. S2 (ESI† le).
with p < 0.05 indicating statistical signicance.
Feasibility of the assay
ESI-MS and MS/MS conditions
Two groups of SMMC-7721 cells in parallel were incubated with
d0
3 3
The ESI-MS and MS/MS data was acquired with an LTQ XL ion- equal molar amounts of Ac GalNAc-a-Bn and Ac GalNAc-a-
d5
trap mass spectrometer equipped with an electrospray ion (ESI) Bn O-glycan precursors (5 Da mass difference) respectively, to
source (Thermo Scientic, USA). The samples were injected into amplify the whole O-glycans. Two groups of the amplied O-
electrospray ion source via a 2 mL Rheodyne loop in a stream of glycans were mixed in 1 : 1 ratio and submitted to per-
ꢁ1
50% methanol at a ow rate of 100 mL min . The spray voltage methylation. Aer direct permethylation, ESI-MS analysis of
d0/d5
was set at 4000 V, with a sheath gas (nitrogen gas) ow rate of 20 Bn
arb, an auxiliary gas (nitrogen gas) ow rate of 5.0 arb, a capil- Bn
-O-glycans was illustrated in Fig. 2A. Ten pairs of the
-O-glycans peaks with signals at m/z 955.25/960.33,
d0/d5
lary voltage of 37 V, a tube lens voltage of 250 V, and a capillary 1200.33/1205.33, 1316.33/1321.33, 1404.42/1409.42, 1561.33/
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of O-glycan peaks with signals at m/z 1316.33/1321.33 and other
nine pairs of O-glycan peaks were achieved by ESI-MS/MS frag-
mentation in Fig. 2B and S3A–I,† correspondingly. According to
d0
the MS data (n ¼ 3), the R values (the intensity ratio of the Bn -
d5
O-glycan to Bn -O-glycan) of each set of peaks were 1.02, 1.05,
0.99, 1.03, 0.92, 1.02, 1.07, 1.07, 0.93 and 0.98, which afforded
an averaged R value of 1.01 that was very close to the theoretical
ratio of 1.0, demonstrating the good feasibility and reliability of
the established method for relative quantitative analysis of O-
glycans in two cells.
Linearity and accuracy verication of the assay
d0/d5
Linearity of ve major pairs of Bn
-O-glycans peaks with
signals at m/z 955.25/960.33, 1200.33/1205.33, 1316.33/1321.33,
+
1
561.33/1566.42 and 1765.42/1770.50 ([M + Na] adducts) were
plotted according to their relative intensities ratios in the
positive mode ESI-MS spectra versus the adding gradient ratios
of O-glycans mixture (10 : 1, 7.5 : 1, 5 : 1, 2.5 : 1, 1 : 1, 1 : 2.5,
1
: 5, 1 : 7.5 and 1 : 10, n ¼ 3), as shown in Fig. 3. The experi-
ment revealed a desirable linear dependency between the
observed relative intensities ratios of ve major peaks of O-
glycans in two groups of SMMC-7721 cells by MS and concen-
tration ratios of the two samples, with the correlation coefficient
being $0.99 in all cases (n ¼ 3, Table 1). The assay clearly
presented a desirable linearity with a 100 folds linear dynamic
range for sample measurements.
d0
Fig. 2 (A) ESI-MS spectra profile of the 1 : 1 mixture of Bn -O-glycans
d5
Clearly, the observed MS intensity ratios were very identical
to the theoretical molar ratios as well as the samples amount
(
blue) and Bn -O-glycans (red) from SMMC-7721 cells and (B)
d0/d5
comparative sequencing of Bn
-O-glycans from SMMC-7721 by
ESI-MS/MS. Each peak was fragmented by CID, respectively. Structure ratios, as listed in Table 1. Moreover, these average ratio data
formulas: yellow square, N-acetylgalactosamine (GalNAc); blue illustrated the averaged CVs (coefficients of variation) values
square, N-acetylglucosamine (GalNAc); yellow circle, galactose (Gal);
(
4.72, 4.07, 4.74, 6.76 and 7.61%, n ¼ 3) lower than 8%,
red triangle, fucose (Fuc); purple diamond, sialic acid (Neu5Ac); gray
diamond, sialic acid (Neu5Gc).
demonstrating the desirable reproducibility and precision of
the established method, aer repeating the experiment in
triplicate.
The accuracy of the method was evaluated by the measured
ratios of d0/d5-Bn-labeled O-glycans versus the theoretical ratios
of d0/d5-Bn-labeled O-glycans to give some relative errors (REs).
1
1
566.42, 1578.17/1583.25, 1649.25/1654.42, 1677.42/1682.33,
765.42/1770.50 and 1939.33/1944.50 ([M + Na] adducts) were
+
observed in MS prole. Structural characterizations of one pair
d0
d5
Fig. 3 (A) The linearity graph of the observed ESI-MS peak abundance ratios of Bn -O-glycans to Bn -O-glycans versus the added ratios of
d0
d5
d0
Bn -O-glycans to Bn -O-glycans and (B) ESI-MS peak abundance ratios of one random pair peaks (m/z 1316.33/1321.33) of Bn -O-glycans to
d5
d0
d5
Bn -O-glycans versus the added ratios of Bn -O-glycans to Bn -O-glycans peaks.
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d0
d5
The mean RE (3.10, 3.99, 2.00, 3.48 and 4.20%, n ¼ 3) was ob- cultured with Ac
3
GalNAc-a-Bn
or Ac
3
GalNAc-a-Bn
tained, as listed in Table 1. Five pairs of the Bn-O-glycans peaks precursor, respectively, and presented the MS-based quantita-
were also presented CV value below 8% and RE value below 5%, tive results as illustrated in Fig. 4. As shown in Fig. 4, 9 and 10
respectively (Table 1, n ¼ 3), which are indicative of rather good Bn-O-glycans were observed in L02 and SMMC-7721 cells,
precision and acceptable accuracy of the established method.
respectively. The characterized structural details of these Bn-O-
glycans were further identied by MS/MS, in consistent with the
previous ref. 29, 37 and 38 One random MS/MS data of the two
peaks with signals at m/z 1043.33 and 1346.33 ([M + Na]
adducts, from L02 cells) was illustrated in Fig. S4.†
ESI-MS combined with MS/MS data has demonstrated that
L02 cells amplied 9 Bn-O-glycans, one neutral Bn-O-glycans
[M + Na] ion peak with signal at m/z 1043.33) and eight
Quali-quantitative proling of Bn-O-glycans between L02 and
SMMC-7721 cells
+
The established IPMAL method has been applied to quali-
quantitatively proling of the repertoire O-glycans from
normal human liver cell line L02 and human hepatoma cell line
SMMC-7721. In parallel, L02 and SMMC-7721 cells were
+
(
Table 1 Linearity, reproducibility and accuracy evaluation of the IPMAL assay
Mixed molar
Measured molar ratios
Mean
Glycan MS
signals (m/z) Regression equation
Correlation
coefficient (R ) glycans
ratios of d0/d5-Bn-labeled of d0/d5-Bn-labeled
reproducibility Mean relative
2
glycans (mean ꢃ SD, n ¼ 3) (%CV, n ¼ 3)
error (%)
9
1
1
1
1
55
Y ¼ 1.0415x ꢁ 0.0399 0.9988
1 : 10
0.105 ꢃ 0.009
0.147 ꢃ 0.008
0.200 ꢃ 0.009
0.409 ꢃ 0.022
1.018 ꢃ 0.043
2.526 ꢃ 0.092
4.992 ꢃ 0.227
7.566 ꢃ 0.327
10.622 ꢃ 0.178
1.106 ꢃ 0.004
0.134 ꢃ 0.016
0.209 ꢃ 0.004
0.418 ꢃ 0.019
1.052 ꢃ 0.008
2.323 ꢃ 0.039
4.798 ꢃ 0.011
7.455 ꢃ 0.357
10.367 ꢃ 0.709
0.093 ꢃ 0.006
0.133 ꢃ 0.011
0.199 ꢃ 0.010
0.406 ꢃ 0.019
0.999 ꢃ 0.013
2.477 ꢃ 0.075
4.874 ꢃ 0.140
7.789 ꢃ 0.519
10.080 ꢃ 0.417
0.093 ꢃ 0.009
0.131 ꢃ 0.006
0.199 ꢃ 0.022
0.393 ꢃ 0.024
0.921 ꢃ 0.045
2.331 ꢃ 0.134
5.225 ꢃ 0.034
7.492 ꢃ 0.584
9.929 ꢃ 1.029
0.095 ꢃ 0.019
0.130 ꢃ 0.013
0.210 ꢃ 0.011
0.394 ꢃ 0.028
0.932 ꢃ 0.104
2.316 ꢃ 0.084
4.702 ꢃ 0.495
7.409 ꢃ 0.311
9.944 ꢃ 0.752
9.04
5.36
4.36
5.28
4.18 (4.72)
3.66
4.56
4.32
1.68
3.66
11.93
2.14
4.66
0.72 (4.07)
1.69
0.23
4.79
6.84
6.58
8.26
5.09
4.65
1.35 (4.74)
3.02
2.87
6.67
4.13
9.37
4.89
10.99
6.19
4.88 (6.76)
5.75
0.64
7.79
10.37
9.25
9.67
5.34
4.89
9.87
0.75
2.31
1.78 (3.10)
1.04
0.17
0.88
6.22
5.86
0.18
4.85
4.43
5.20 (3.99)
7.07
4.04
0.60
3.67
7.04
0.56
0.71
1.55
0.04 (2.00)
0.91
2.52
3.85
0.80
7.28
1.70
0.69
1.68
7.86 (3.48)
6.76
4.50
0.11
0.71
1
1
1
1
2
5
7
1
: 7.5
: 5
: 2.5
: 1
.5 : 1
: 1
.5 : 1
0 : 1
200
316
561
765
Y ¼ 1.017x ꢁ 0.047
0.9984
1 : 10
1
1
1
1
2
5
7
1
: 7.5
: 5
: 2.5
: 1
.5 : 1
: 1
.5 : 1
0 : 1
Y ¼ 1.015x ꢁ 0.0201
0.9993
1 : 10
1
1
1
1
2
5
7
1
: 7.5
: 5
: 2.5
: 1
.5 : 1
: 1
.5 : 1
0 : 1
Y ¼ 1.0018x ꢁ 0.0182 0.9992
1 : 10
1
1
1
1
2
5
7
1
: 7.5
: 5
: 2.5
: 1
.5 : 1
: 1
.5 : 1
0 : 1
Y ¼ 0.9892x ꢁ 0.0454 0.9993
1 : 10
7.14
2.27
5.09
1.45
6.76 (4.20)
7.37
5.95
1.22
0.56
1
1
1
1
2
5
7
1
: 7.5
: 5
: 2.5
: 1
.5 : 1
: 1
.5 : 1
0 : 1
7.17
11.17 (7.61)
3.63
10.53
4.19
7.57
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those of L02 cells. Fucosylated core 2 O-glycans were up-
regulated in SMMC-7721 cells. None of neutral O-glycan was
observed in SMMC-7721 cells. Data indicated that SMMC-7721
express more O-glycans and higher levels of sialylated and/or
fucosylated O-glycans over that of normal cell line L02, in line
with the facts that abnormal sialylated and fucosylated O-
glycans in biological processes were closely related to the
35
occurrence, development and deterioration of liver tumor.
Quali-quantitative proling and distinction of both neutral
and sialylated Bn-O-glycans between L02 and SMMC-7721
cells
Due to the biological importance of the glycan terminal sialy-
lation associated with infection or disease development, it is
important to establish novel and effective method for linkage-
specic distinction of sialic acids. Despite sialylated Bn-O-
glycans can be successfully determined by ESI-MS, it is very
challenging to assign sialic acids specic linkages by ESI-MS
and/or MS/MS, as presented in Fig. 2B, S3 and S4 (ESI† le).
Fortunately, a linkage-specic assignation of O-glycans terminal
Fig. 4 (A) Quali-quantitative profiling of Bn-O-glycans between L02 sialic acid could be achieved by combining our IPMAL assay
and SMMC-7721 cells by ESI-MS and (B) bar charts derived from the with sialic acid derivatization (a mild dimethylamidation and
relative intensities of the MS signals in (A). The intensity ratio of the
Bn -O-glycans to its corresponding Bn -O-glycans is indicated as R
permethylation), which could selectively convert a2,6-linked
sialic acids to dimethylamidation and a2,3-linked sialic acid to
lactones, as shown in Fig. S2.†
d0
d5
(
n ¼ 3, averaged). Note: *p < 0.05, **p < 0.01. Structure formulas:
yellow square, N-acetylgalactosamine (GalNAc, N); blue square, N-
acetylglucosamine (GlcNAc, N); yellow circle, galactose (Gal, H); red
To ensure the feasibility of the assay, standard sialylated
triangle, fucose (Fuc, F); purple diamond, sialic acid (Neu5Ac, S); white trisaccharides, a2,3-Neu5Ac-Lac (3 -SL) and a2,6-Neu5Ac-Lac
0
gray diamond, sialic acid (Neu5Gc, G).
0
(
6 -SL), were submitted to the sialic acids specic derivatiza-
tion and proled by ESI-MS, as shown in Fig. S5 (ESI† le).
sialylated Bn-O-glycans ([M + Na] adducts with signals at m/z According to the structural changes of glycans with sialic acid
+
955.25, 1200.33, 1316.33, 1346.33, 1404.42, 1578.17, 1765.42 ends aer the specic derivatization reactions, a mass differ-
and 1939.33), as shown in Fig. 4. Whereas, SMMC-7721 ence (13 Da) between a2,3- and a2,6-sialic acid can be detected,
+
expressed 10 Bn-O-glycans ([M + Na] adducts with signals at facilitating the isomeric assignment of a2,3- and/or a2,6-sialy-
+
m/z 960.33, 1205.33, 1321.33, 1409.42, 1566.42, 1583.25, lated glycans. As we expected, distinct [M + Na] ionized peaks at
0
0
1
654.42, 1682.33, 1770.50 and 1944.50), which were either high m/z 838 and 851 (13 Da) were assigned to be 3 -SL and 6 -SL,
sialylated or fucosylated ones as later conrmed by MS/MS. In respectively, demonstrating the feasibility of the assay for
Fig. 4, seven pairs of O-glycan peaks with a 5 Da difference linkage specic characterization of glycan isomers with a2,3-
including m/z 955.25/960.33, 1200.33/1205.33, 1316.33/1321.33, and a2,6-sialic acid ends.
1
1
404.42/1409.42, 1578.17/1583.25, 1765.42/1770.50 and
We further applied our IPMAL method combined with sialic
939.33/1944.50, were observed in MS prole. From relative acids specic derivatization to achieve quali-quantitative
intensities signals in MS, the relative quantitation of these O- proling and distinction of both neutral and sialylated Bn-O-
glycans in L02 and 7721 cells were indicated to be R ¼ 1.26, 0.29, glycans between L02 and SMMC-7721 cells. Aer combining the
0.70, 2.72, 0.88, 0.96 and 0.31. Two single peaks with signals at IPAML assay with sialic acid derivatization, not only the sialic
m/z 1043.33 and 1346.33 were detected in L02 only, whereas acids residues at O-glycans in L02 and SMMC-7721 were further
three single O-glycan peaks with signals at m/z 1566.42, 1654.42 stabilized, but also the linkage specicity as well as the relative
and 1682.33 with high sialylation level, were observed in SMMC- quantitation could be well characterized, as shown in Fig. 5.
7721 cells only. Furthermore, based on relative abundance
Compared with the relative intensity of Bn-O-glycans in L02
values of each pair of O-glycans peaks in MS, relative quanti- and SMMC-7721 (Fig. 4A) in MS, MS signals at m/z 955.33/
cation of O-glycans between normal and tumor cells was ach- 960.33, 1043.42, 1200.50/1205.42, 1316.42/1321.42, 1404.42/
ieved, as illustrated in Fig. 4B.
1409.42, 1566.50, 1654.42, 1765.58/1770.58 and 1939.67/
+
As shown in Fig. 4, O-glycans in SMMC-7721 cells with sugar 1944.67 ([M + Na] ion peaks) did not change, indicating the
compositions of N2H1S1, N1H1S2, N2H2F1S2 (molecular ion presence of neutral or sialylated Bn-O-glycans with a2,3-linked
+
peaks [M + Na] with signals at m/z 1205.33, 1321.33 and sialic acid (Neu5Ac), as shown in Fig. 5A. Whereas, Bn-O-glycans
1
944.50) were signicantly higher than those of L02 cells. with a2,6-linked sialic acid (Neu5Ac) terminals yielded a 13 Da
+
Moreover, N2H2F1S1 and N2H2S2 ([M + Na] ion peaks with mass increase. For example, peaks at m/z 955.25/960.33,
signals at m/z 1583.25 and 1770.50) was slightly higher than 1316.33/1321.33, 1566.42 and 1682.33, in Fig. 4A were shied
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“light” and “heavy” O-glycan precursors could be chemically
synthesized by one-step O-glycosylation, which shows method-
ological advantage over previous work and will make easy
generalization and wide application of the established assay.
Through metabolic oligosaccharide engineering and isotopic
labeling, the MS-based quali-quantitative proling of Bn-O-
glycans derived from two cells could be achieved simulta-
neously through the mass differences (5 Da) in pairs of peaks in
MS. The quantitation feasibility, reproducibility, reliability and
linearity of this strategy were fully investigated, yielding 100 fold
linear dynamic range, good reproducibility (CV, 4.07–7.61%)
and accuracy (RE, 2–4.2%). The quali-quantitative determina-
tion of Bn-O-glycans from L02 and SMMC-7721, were achieved
successfully, which is benecial for the mechanism elucidation
of O-glycosylation as required in clinical and pathological
research.
When the IPAML approach was combined with sialic acid
specic derivatization, quali-quantitative determination of
neutral and sialylated O-glycans terminal sialic acids with a2,3-
and/a2,6-linkages based on MS prole could be accomplished
Fig. 5 (A) Quali-quantitative profiling and distinction of both neutral the rst time. The proposed assay has been successfully applied
and sialylated Bn-O-glycans between L02 and SMMC-7721 cells and to quali-quantitative proling and sialic acid linkage specic
(
(
B) bar charts derived from the relative intensities of the MS signals in
discrimination of the whole O-glycans derived from L02 and
SMMC-7721 cell. As the total sialic acids expression level as well
as their linkage specicity could be used to predict the risk of
d0
A). The intensity ratio of the Bn -O-glycans to its corresponding
d5
Bn -O-glycans is indicated as R (n ¼ 3, averaged). Notes: *p < 0.05;
*
*p < 0.01. Structure formulas: yellow square, N-acetylgalactosamine
(
GalNAc); blue square, N-acetylglucosamine (GlcNAc); yellow circle, various diseases such as tumor, the proposed assay which
galactose (Gal); red triangle, fucose (Fuc); gray diamond, a2,3- facilitates quali-quantitatively proling the whole O-glycans
Neu5Gc; pink diamond, a2,3-Neu5Ac; green diamond, a2,6-Neu5Ac;
purple diamond, a2,8-Neu5Ac.
especially sialylated ones, shows great potential for clinical
biomarker discovery.
In addition, the method with the merits of bypassing the
to m/z 968.33/973.42, 1329.42/1334.42, 1579.50 and 1695.50 in
releasing and relabeling of the glycan, affording the Mucin-type
Fig. 5A, suggesting the presence of a2,6-sialylated O-glycans. MS
signal at m/z 1359.33 was assigned to be O-glycan with a a2,3-
linked sialic acid (Neu5Gc) and a a2,6-linked sialic acid
O-glycan itself with chromophore group, which not only
improves the detection sensitivity of mass spectrometry, but
also enables the further HPLC and LC-MS separation analysis.
(Neu5Ac). Structural details of all other peaks, including
The proposed method can also be applied to comprehensive
screening meaningful O-glycans from clinical samples such as
tissue.
The method is effective and highly reproducible. With all the
merits described above, we believe this method will make
signicant contribution to glycomics.
The excellent applicability of the suggested procedure was
validated via quantitative comparison of the O-glycans of perch
and salmon eggs by ESI-MS and RP-HPLC-UV-ESI-MS/MS.
neutral, di- and tri-sialylated O-glycans have been determined
by MS/MS, providing the proposed information of O-glycans
terminal sialic acids linkage specicity, as shown in Fig. 5A. The
quali-quantitative proling and distinction of both neutral and
sialylated Bn-O-glycans with linkage-specicity was indicated in
Fig. 5B. As shown in Fig. 5B, 8 O-glycans were up-expressed in
SMMC-7721 cells, while 4 O-glycans were down-expressed in L02
cells. Moreover, most of the up-expressed O-glycans are highly
sialylated. The expression level of specic O-glycans such as the
disialyl core 1 Neu5Ac-a-2,3-Gal-b-1,3-(Neu5Ac-a-2,6)-GalNAc-a-
O-Thr in SMMC-7721 cells was higher over that of L02 cells, in
Conflicts of interest
8,35
line with the previous works.
Therefore, the good compati-
There are no conicts to declare.
bility of our IPMAL assay with sialic acid derivatization (a mild
dimethylamidation and permethylation), shows great potential
in quali-quantitative determination of the O-glycans with or
without sialic acids between normal and disease.
Acknowledgements
This work was funded by National Natural Science Foundation
(No. 31300678, 31370804, 21375103 and 31670808), the Scien-
tic Research Program Fund for Shaanxi Province Key Labora-
tory (14JS101), Shaanxi Basic Research Program of Natural
Conclusions
An innovative and effective approach termed as IPMAL for Science (2016JQ3018), Outstanding Youth Backbone of North-
quali-quantitative proling of neutral and sialylated Bn-O- west University and Graduate Innovation Fund (YZZ17163 and
glycans by ESI-MS in positive-ion mode has been developed. The YZZ15067).
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1
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