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Utilizing Sub-ambient Pressure Ionization with Nanoelectrospray (SPIN) to Enable High Sensitivity Detection and Extended Structural Coverage of N-glycans in Human Serum Jonathan T Cox, Scott R. Kronewitter, Anil K. Shukla, Ronald J. Moore, Keqi Tang and Richard D. Smith Biological Sciences Division, Pacific Northwest National Laboratory
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www.omics.pnl.gov CONTACT: Keqi Tang, Ph.D. Biological Sciences Division Pacific Northwest National Laboratory E-mail: keqi.tang@pnnl.gov
Acknowledgements Portions of this research were supported by the National Cancer Institute (1R33CA155252), the NIH National Institute of General Medical Sciences (8 P41 GM103493-10) and the U.S. Department of Energy (DOE) Office of Biological and Environmental Research (OBER) Pan-omics program at Pacific Northwest National Laboratory (PNNL) and performed in the Environmental Molecular Sciences Laboratory, a DOE OBER national scientific user facility on the PNNL campus. High performance computing research was performed using PNNL Institutional Computing. PNNL is a multi-program national laboratory operated by Battelle for the DOE under contract DE-AC05-76RL01830.
Overview The glycan profiling of human serum by mass spectrometry has been employed to study several disease conditions and demonstrated promise in clinical biomarker discovery. However, investigations into the extent of glycosylation of human serum proteins is currently limited by the quality of instrumental analysis. Larger glycans, such as heavily sialylated and polysialylated glycans are typically not detected by LC-MS because they are extremely labile and also exhibit low ionization efficiency. By combining subambient pressure ionization with nanoelectrospray (SPIN) coupled to high resolution MS and advanced data processing tools, we demonstrate much extended glycan and sialic acid polymer coverage as compared to a conventional ESI-MS interface. The benefits of improved instrument sensitivity and gentler ionization conditions in the SPIN-MS interface facilitate the detection of sialic acid containing N-glycans without the need of sample derivatization. In addition, we show that SPIN-MS is effective in elucidating structures of heavily sialylated and polysialylated glycans previously unattainable by ESI.
Conclusions • The SPIN source enables more sensitive
MS detection and identification of structural features not observed with a conventional ESI source.
• The SPIN-MS interface provides gentler ionization conditions and significantly reduces in-source fragmentation.
• Combining advancements in sample preparation, high resolution mass spectrometry, and data processing enabled the elucidation of large families of polysialylated N-glycans in human serum.
References 1. Cox, J. T.; Kronewitter, S. R.; Shukla, A. K.; Moore, R.
J.; Smith, R. D.; Tang, K. Anal. Chem. 2014, 86 (19). pp 9504-9511
2. Kronewitter, S. R.; Marginean, I.; Cox, J. T.; Zhao, R.; Hagler, C. D.; Shukla, A. K.; Carlson, T. S.; Adkins, J. N.; Camp, D. G.; Moore, R. J.; Rodland, K. D., and Richard D. Smith. Anal. Chem. 2014, 86 (17), pp 8700-8710
3. Marginean, I.; Page, J. S.; Tolmachev, A. V.; Tang, K.; Smith, R. D. Anal. Chem. 2010, 82, pp 9344-9349.
4. Kronewitter, S. R.; Slysz, G. W.; Marginean, I.; Hagler, C. D.; LaMarche, B. L.; Zhao, R.; Harris, M. Y.; Monroe, M. E.; Polyukh, C. A.; Crowell, K. L.; Fillmore, T. L.; Carlson, T. S.; Camp, D. G.; Moore, R. J.; Payne, S. H.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2014, 86 (13), pp 6268-6276
Methods Experimental
LC-MS
Data Processing
Structural Elucidation
• Agilent Nanoflow LC (1260 Series) • 75 cm long, 75 µm i.d., 360 µm o.d. columns
packed with 3 µm Hypercarb particles • Thermo Exactive Orbitrap equipped with either a
SPIN source or an ESI source
• GlyQ-IQ HPC software package • Windows server 2012 HPC 1504 cores
• Models and fits: • Isotope profiles • Extracted ion chromatograms
• Theoretical target library
• Exact mass analysis • GlycoGrid Profile Visualization
Results Reduced in-source fragmentation Increased Glycan Coverage
Instrument Configuration
ESI-MS vs. SPIN-MS : Mass spectra of a N-glycan containing sialic acid, Hex5HexNAc4NeuAc2 (2224.01 Da), from the LC-MS analysis of human serum obtained from the conventional ESI-MS interface operated with capillary inlet temperatures of 300 °C , and 150 °C, and for the SPIN interface. The red arrows represent the 3+ and 2+ charge states of the observed intact glycan.
.
400 600 800 1000 1200 1400 0.0
6.0x107
1.2x107
1.8x107
2.4x107
m/z
366.14
657.24
528.19 819.29
2+
1113.39 292.10
742.60
400 600 800 1000 1200 1400 0.0
3.0x107
6.0x107
9.0x107
1.2x108
m/z
1113.39
3+
2+
967.85
400 600 800 1000 1200 1400 0.0
3.0x106
6.0x106
9.0x106
1.2x107
m/z
292.10
657.24
819.29
1113.39
742.60
3+
2+
366.14
967.85
967.85 454.15 893.32
Inte
nsity
(a.u
.) In
tens
ity (a
.u.)
Inte
nsity
(a.u
.)
ESI 300 °C
ESI 150 °C
SPIN
x 1
03 x
103
x 1
03 x
103
x 1
03 x
103
x 1
03 x
103
Polysialic Acid Family (Hex6-HexNAc7-Fuc1-2-NeuAc7-11)
x 1
03
x 1
03
SPIN Exactive (with Polysialic acid Previous Library (without Polysialic acid)
Fuco
se
HexNAc2 HexNAc3 HexNAc4 HexNAc5 HexNAc6 HexNAc7 HexNAc8
Hex 3
Hex 4
Hex 5
Hex 6
Hex 7
Hex 8
Hex 9
Hex 10
Polysialic Acid
Region
Sialic Acid
0-15 0-7
Human Blood Serum Glycan Profile: This matrix shows the compositions of all the glycans detected in human serum. The columns correspond to the number of HexNAc monosaccharides and the rows correspond to the number of Hex monosaccharides. The detailed axes within each group depicts degree of fucosylation (y-axis) and sialylation (x-axis) present.
Polysialylated N-Glycan Family: Example mass spectra representing a family of 10 polysialylated N-glycans. Each member differed by another by either a fucose or sialic acid.
Schematic of the SPIN-MS interface: The standard interface ion optics in front of lens L0 are replaced by a dual ion funnel interface and the electrospray emitter is placed inside the first vacuum region of the mass spectrometer in front of the first (high pressure) ion funnel.
Sample Preparation
• N-Glycan release, Microwave Reactor • Sodium Borohydride Reduction • Solid Phase Purification (C8/Graphite)
C - trap
Transfer Multipole
Orbitrap Mass Analyzer
Low Pressure Ion Funnel
High Pressure Ion Funnel
~ 2 Torr ~ 20 Torr 760 Torr
From LC
Sheath Gas
Emitter
800 600
400 200
0 1536 1535 1534 1533
300
200
100
0 1226 1225 1224 1223
6 - 7 - 1 - 07
6 - 7 - 1 - 08
80 60 40 20 0
1730 1729 1728 1727
6 - 7 - 1 - 09
120
80
40
0 1371 1370 1369 1368
6 - 7 - 1 - 10
12
8
4
0 1924 1923 1922 1921
6 - 7 - 1 - 11
500 400 300 200 100
0 1189 1188 1187 1186
6 - 7 - 2 - 07
400
300
200
100
0 1262 1261 1260 1259
6 - 7 - 2 - 08
800
600 400
200
0 1335 1334 1333 1332
6 - 7 - 2 - 09
600
400
200
0 1408 1407 1406 1405
30
20
10
0 1481 1480 1479 1478
6 - 7 - 2 - 11
4+
6 - 7 - 2 - 10
4+
3+
3+
4 +
4+
4+ 4+
4+ 3 + x7 x7
x8 x8
x9 x9
x10 x10
x11 x11
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