Quantitation of Heparan Sulfate: A Comparison of UPLC-HILIC- and PGC-MS/MS using Multiple Reaction Monitoring
Jing Li, Elaine Stephens, and Mario DiPaola
Blue Stream Laboratories, 763 Concord Ave, Bldg E, Cambridge, MA 02138 [email protected] 617-234-0001
ABSTRACT
MATERIALS AND METHODS
RESULTS
RESULTS
08/08/2013 23:29:42
Figure 3. Analysis of Disaccharide Standards in Human
Serum by UPLC-HILICMS/MS
RT:
0.0 - 31.0
7dUA_500ng_IS1ug_08Aug13_1
50
0
100
50
S_I_III_III-A_I_III_IV-S_500ng_IV-A_5...
50
50
50
0
5
0
100
IS: 354.2 113.0
12
10
5
15
Time (min)
y = 0.0210x - 0.0664
R² = 0.9963
8
6
4
2
0
-2 0
100
10
15
20
25
Time (min)
30
35
40
I-A: 268.7 97.0
NL: 7.88E2
5
5
5
10
TIC F: - cHILIC-MS/MS
ESI SRM ms2 268.650
[96.999-97.001]
MS
ICIS 7dUA_500ng_IS1ug_08Aug13_1
5
10
10
IV-S: 416.0 138.0
NL: 1.31E4
TIC F: - c ESI SRM ms2 416.000 [137.999-138.001]
MS ICIS 7dUA_500ng_IS1ug_08Aug13_1
Table 2. Concentrations of Disaccharides Derived from Heparan Sulfate in
NL: 3.16E3
TIC F: - Human
c ESI SRM
ms2
247.650
[96.999-97.001]
MS Determined using UPLC-HILICSerum
and
Cerebrospinal
Fluid (CSF)
ICIS 7dUA_500ng_IS1ug_08Aug13_1
I-S: 287.5  97.0
NL: 7.99E2
TIC F: - c ESI SRM ms2 287.450 [96.999-97.001] MS
Concentration
ICIS 7dUA_500ng_IS1ug_08Aug13_1
IS: 354.2 113.0
NL: 1.06E3
(ng/mL)
TIC F: - c ESI SRM ms2 354.200 [112.999-113.001]
MS ICISNormal
7dUA_500ng_IS1ug_08Aug13_1
Human Serum
20
25
200
300
400
500
4
15
10
5
0
-5
0
100
200
300
400
500
Normalized Peak Area
20
0
0
100
20
10
0
100
200
500
2
400
500
1.5
1
0.5
0
-0.5 0
100
200
300
III-S
y = 0.0924x - 0.6819
R² = 0.9926
0
400
Concentration (ng/mL)
30
-10
300
y = 0.0056x - 0.0776
R² = 0.9971
IV-S
40
200
I-A
2.5
Concentration (ng/mL)
50
300
400
500
Concentration (ng/mL)
Normal Human CSF
(Blue Stream)
137
Normal Human CSF
(Literature2)
8
7
6
5
4
3
2
1
0
-1 0
III-A
II-A
I-A
7
IV-S
III-S
I-S
18
25
11
22
140 (Total)
Pooled normal human serum (100 µL) or CSF (100 µL) was spiked with 100 ng internal standard and
digested with heparinase I, II and III. The digestion was cleaned up by Amicon 3K centrifugal filter unit
followed by Hypercarb SPE column. The eluate was dried, resolubilized in 100 µL of 90% acetonitrile in
water, and injected onto a HILIC column (Waters Acquity UPLC BEH glycan column, 50 mm x 2.1 mm, 1.7
µm particle size). The column eluate was analyzed by a triple quadrupole mass spectrometer (Thermo TSQ
Vantage) using multiple reaction monitoring (MRM) in negative ion mode. The concentration of each
disaccharide in the serum or CSF was calculated from the calibration curves shown in Figure 4.
CONCLUSIONS
1
3
y = 0.0465x - 0.2494
R² = 0.9970
98
2
-1
113
Normal Human Serum
(Literature1)
y = 0.0063x - 0.0609
R² = 0.9941
3
Concentration (ng/mL)
25
(Blue Stream)
30
III-A
Concentration (ng/mL)
0
0
10
IV-A
Normalized Peak Area
I-S: 287.5  97.0
100
Figure 4. Calibration Curves of Disaccharide Standards in Human Serum
Determined by UPLC-HILIC-MS/MS
IV-S: 416.0 138.0
0
100
50
Representative MRM chromatograms of disaccharide standards analyzed by HILIC-MS/MS. Known
concentrations of disaccharides standards and internal standard (IS) were spiked in human serum. The mixture
was cleaned up by Amicon 3K centrifugal filter unit followed by Hypercarb SPE column. The disaccharides
fraction was dried, reconstituted in 90% acetonitrile in water, and injected onto a HILIC column (Waters Acquity
UPLC BEH glycan column, 50 mm x 2.1 mm, 1.7 µm particle size). The column eluate was analyzed by a triple
quadrupole mass spectrometer (Thermo TSQ Vantage) using multiple reaction monitoring (MRM) in negative ion
mode. Transitions that are specific to each disaccharide were monitored and shown in the figure. Disaccharide IA and I-S are prone to in-source fragmentation. Therefore, they were observed in III-A/II-A and III-S
chromatograms, respectively. Anomers of the disaccharides were resolved by HILIC column and observed as
two peaks in the chromatograms.
0
100
III-S: 247.5  97.0
5
0
I-A: 268.7 97.0
0
100
50
IV-A
50
Normalized Peak Area
0
100
II-A: 458.0 300.0
NL: 8.10E3
PGC-MS/MS
10
25
10
TIC F: - c ESI SRM ms2 458.004 [299.999-300.001]
MS ICIS 7dUA_500ng_IS1ug_08Aug13_1
0
100
This work describes the development of an analytical method to quantify heparan
sulfate in serum and other biological matrices using a triple quadrupole mass
spectrometer set-up in multiple reaction monitoring in negative ion mode. Seven
commercially purchased disaccharides standards from HS were evaluated using
two LC-MS/MS methods, HILIC-MS/MS and PGC-MS/MS. The HILIC-MS/MS
produced superior chromatographic profiles resulted in a more robust and
sensitive method. Excellent linearity between 1 ng/mL to 500 ng/mL and LOQ
levels as low as 5 ng/mL were obtained for most disaccharides. The HILIC-MS/MS
method was then applied to analyze disaccharides derived from the heparin lyase
digestion of HS in 0.1 mL of normal human serum or CSF. With proper sample
preparation, purification and calibration with internal standard, accurate
quantification of the serum and CSF disaccharides was achieved. The analytical
method developed herein is suitable for the accurate quantitation of HS derived
from a variety of biological matrices, which would have significant applications in
biomarker discovery and disease diagnosis.
y = 0.0151x - 0.2992
R² = 0.9896
REFERENCES
1
100
200
300
400
500
Concentration (ng/mL)
T. Oguma, S. Tomatsu, A. M. Montano and O. Okazaki; Analytical Biochemistry
2007, 368: 79-86.
2 H. Zhang, S. P. Young, C. Auray-Blais, P. J. Orchard, J. Tolar and D. S.
Millington; Clinical Chemistry 2011 57:1005-1012.
45
Representative MRM chromatograms of disaccharide standards analyzed by PGC-MS/MS. Known
concentrations of disaccharides standards and internal standard (IS) were spiked in human serum. The mixture
was cleaned up by Amicon 3K centrifugal filter unit. The flow-through was dried, reconstituted in 0.1 mL water
and injected onto a PGC column (Thermo Hypercarb, 100 mm x 2.1 mm, 5 µm particle size). The column eluate
was analyzed by a triple quadrupole mass spectrometer (Thermo TSQ Vantage) using multiple reaction
monitoring (MRM) in negative ion mode. Transitions that are specific to each disaccharide were monitored and
shown in the figure. Disaccharide I-A and I-S are prone to in-source fragmentation. Therefore, they were
observed in III-A/II-A and III-S chromatograms, respectively.
I-S
Normalized Peak Area
Relative Abundance
II-A: 458.0 300.0
I-S
MS/MS are Comparable with Literature Values
II-A
0
100
III-S
III-S: 247.5  97.0
0
100
RT: 0.0 - 47.0 SM: 7B
100
III-A: 458.0 237.0
IV-S
0
100
Figure 2. Analysis of Disaccharide Standards in Human Serum by PGC-MS/MS
0
100
I-A
0
100
02/08/2013 06:55:18
IV-A: 378.1 175.0
III-A: 458.0 237.0
NL: 8.04E2
LOQ (ng/mL)
IV-A
III-A
II-A
TIC F: - c ESI SRM ms2 458.006 [236.999-237.001]
MS ICIS 7dUA_500ng_IS1ug_08Aug13_1
0
100
Normalized Peak Area
Liquid Chromatography-Mass Spectrometry
PGC-LC: The disaccharide samples were separated using a Hypercarb column
(Thermo Scientific, 2.1 mm ID x 100 mm, 5 µm). The column temperature was room
temperature. Solvent A was 10 mM ammonium bicarbonate (pH 10) and solvent B
was acetonitrile. The gradient was 0% B for 3 mins, linearly modified to 20% B over
40 mins, modified to 90% B in 0.5 min, maintained at 90% B for 3.5 mins, modified
to 0% B in 1 mins, and finally maintained at 0% B for 12 mins. The flow rate was 0.2
mL/min.
HILIC-LC: The disaccharide samples were separated using a HILIC column (Waters
Acquity UPLC BEH glycan column, 2.1 mm ID x 50 mm, 1.7 µm). The column
temperature was room temperature. Solvent A was 12.5 mM ammonium formate
(pH 4.4) and solvent B was solvent A in 90% acetonitrile. The gradient was 0% A for
2 mins, linearly modified to 15% A over 4 mins, then linearly modified to 35% A
over 20 mins, maintained at 35% A for 5 mins, modified to 0% A over 0.1 min, and
finally maintained at 0% A for 14 mins. The flow rate was 0.2 mL/min.
MS Settings: A Thermo TSQ Vantage Triple Quadrupole mass spectrometer
equipped with a standard Ion Max source was used. The mass spectrometer was
operating in multiple reaction monitoring in negative ion mode. The capillary
temperature was set at 250 ºC, and spray voltage was set at -2500 V. The sheath
gas pressure was 35 psi, and auxiliary gas pressure was 10 psi. A specific and
sensitive transition was choosen for each disaccharide. The S-lens and collision
energy were optimized for each transitions respectively.
TIC F: - c ESI SRM ms2 378.100 [174.999-175.001]
MS ICIS 7dUA_500ng_IS1ug_08Aug13_1
0
100
50
RESULTS
Figure 1. Structures of Disaccharides that Constitute the Major Building
Blocks of Heparan Sulfate Chains
IV-A: 378.1 175.0
50
Normalized Peak Area
Materials
Heparan sulfate derived disaccharide standards IV-A, III-A, II-A, I-A, IV-S, III-S, I-S
were purchased from V-labs (Covington, LA). Internal standard chondrosine was
purchased from GlycoSyn (Raleigh, NC). Heparinase I, heparinase II and
heparinase III from Flavobacterium heparinum were purchased from Syd Labs
(Malden, MA). Pooled normal human serum and CSF were purchased from
BioChemed (Winchester, VA).
100
Normalized Peak Area
MATERIALS AND METHODS
Sample Preparation
A sample of 0.1 mL human serum or CSF was spiked with 100 ng internal standard
and treated with 4 mU each of heparinases I, II and III in digestion buffer (50 mM
NH4OAc, 1 mM Ca(OAc)2, pH 8.0) in an Amicon 3K centrifugal filter unit. The
sample was placed in a water bath at 37 ºC for 24 hours and centrifuged at 8000 g
for 15 minutes. 0.1 mL of water was added to the filter unit and the sample was
centrifuged at 8000 g for 15 minutes. For PGC-MS/MS, the combined flow-through
containing disaccharides and internal standard was dried, reconstituted in 0.1 mL
water and analyzed by LC-MS. For HILIC-MS/MS, the combined flow-through was
purified by Hypercarb SPE column in accordance with manufacture’s instructions.
The disaccharides fraction was dried, reconstituted in 0.1 mL 90% acetonitrile in
water, and analyzed by LC-MS.
Relative Abundance
Heparan sulfate (HS) is a linear polysaccharide found in the extracellular matrix and
biological fluids of animals where it interacts with hundreds of proteins and performs
a variety of critical roles. Many biological functions can be monitored directly by
their impact on HS quantity; therefore, sensitive and robust quantitative HS
methods are critical in order to accurately monitor these processes. This work
describes the development of a robust and sensitive method to quantify HS in
serum and other biological matrices using a triple quadrupole mass spectrometer
(Thermo TSQ Vantage) set-up in multiple reaction monitoring (MRM) in negative ion
mode. Quantitative methods were initially evaluated using disaccharide HS
standards which were directly analyzed HILIC-MS/MS (hydrophilic interaction
chromatography MS/MS, 2.1 mm I.D. UPLC BEH glycan column (Waters), 1.7 µm
particle size) and PGC-MS/MS (2.1 mm I.D. Porous graphitic carbon, Hypercarb
column (Thermo)). Seven commercially purchased disaccharides standards from
HS were analyzed within the range of 1ng/mL to 500 ng/mL. Excellent sensitivity
and linearity was obtained using UPLC-HILIC-MS/MS by which it was possible to
resolve all seven disaccharides. The resulting sharp peaks allowed for LOQ levels
as low as 5 ng/mL for most disaccharides. By comparison, the hypercarb column
gave broader peaks in the chromatographic profile, but was still able to resolve all
seven HS disaccharide standards. Due to the lower resolution, this PGC-MS/MS
method exhibited a higher LOQ of between 5-100 ng/mL. The HILIC-MS/MS
method was then applied to analyze disaccharides derived from the heparin lyase
digestion of HS in 0.1 mL of normal human serum or cerebrospinal fluid (CSF)
spiked with an internal standard to correct for losses during sample preparation,
which allowed for accurate quantification of the serum and CSF disaccharides. The
superior chromatographic profile from UPLC-HILIC-MS/MS resulted in a more
robust and sensitive method, suitable for the accurate quantitation of HS derived
from a variety of biological matrices.
Table 1. Comparison of the Limit of Quantification (LOQ) of Disaccharide
Standards in Human Serum Analyzed using PGC-MS/MS and UPLC-HILICNL: 5.94E3
MS/MS
Normalized peak area = Peak area of the analyte /
peak area of the internal standard. Calibration
curves were constructed by plotting normalized
peak areas against the concentration of the
analytes. The calibration curves for the seven
disaccharide standards evaluated were linear over
the concentration range of 1 ng/mL to 500 ng/mL.
The correlation coefficients (R2) were not less than
0.9896.
0.5
y = 0.0009x - 0.0190
R² = 0.9936
0.4
0.3
0.2
0.1
0.0
-0.1
0
100
200
300
Concentration (ng/mL)
400
500
ACKNOWLEDGMENTS
We would like to acknowledge all the members of Blue Stream Laboratories for
their support.
The work described herein is intended for research purposes only; not intended for any human or animal
therapeutic or diagnostic use, unless otherwise stated. The trademarks or copyrights mentioned herein are the
property of their respective owners.